Antibodies that neutralize rsv, mpv and pvm and uses thereof

ABSTRACT

The invention relates to antibodies, and antigen binding fragments thereof, that neutralize infection of both RSV, MPV and PVM. The invention also relates to nucleic acids that encode, immortalized B cells and cultured plasma cells that produce, and to polypeptides that bind to such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies, antibody fragments, and polypeptides recognized by the antibodies of the invention in screening methods as well as in the diagnosis, treatment and prevention of RSV or MPV infection and RSV and MPV co-infection.

This application claims the benefit of priority of U.S. provisionalApplication No. 61/613,197, filed Mar. 20, 2012, and U.S. provisionalApplication No. 61/655,310, filed Jun. 4, 2012, the disclosures of whichare hereby incorporated by reference, as if written herein, in theirentireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 470082_401C2 SEQUENCE LISTING.txt. The text fileis 25.4 KB, was created on May 27, 2020, and is being submittedelectronically via EFS-Web.

BACKGROUND

Respiratory Syncytial Virus (RSV) and Metapneumovirus (MPV) andPneumonia Virus of mice are common cold viruses belonging to the familyof paramyxovirus that share target population and represent a majorhealth problem in newborns and immunocompromised patients.

RSV is the major cause of acute respiratory tract disease in infants andadults across the globe. Between 0.5% and 3.2% of children with RSVinfection require hospitalization (Thompson, W. W. et al., 2003, JAMA:The Journal of the American Medical Association 289:179-186), and 5% to10% of children have prolonged severe infection, a factor believed to bepredisposing to wheezing and asthma-like symptoms later in childhood.Immunity to RSV appears to be short-lived, thus re-infections arefrequent (Ogra, 2003, Paediatric Respiratory Reviews 5 SupplA:S119-126).

The human MPV was isolated for the first time in 2001 and is nowrecognized to be the second major cause of acute respiratory tractdisease in infants and adults; it is estimated that it infects over 50%of infants by two years of age and almost all children by five years.MPV accounts for roughly 5 to 15% of respiratory disease in hospitalizedyoung children (Alto, 2004, The Journal of the American Board of FamilyPractice/American Board of Family Practice 17:466-469; Williams et al.,2004, N Engl J Med 350:443-450). Infection with MPV is a significantburden of disease in at-risk premature infants, chronic lung disease ofprematurity, congestive heart disease, and immunodeficiency (Martino etal., 2005, Biology of Blood and Marrow Transplantation: Journal of theAmerican Society for Blood and Marrow Transplantation 11:781-796).

Co-infections with MPV and RSV may be common given their prevalence andoverlapping winter epidemics. Although it is unclear whether synergisticpathology can occur between these two viruses, exacerbations leading toparticularly severe respiratory tract disease were observed in somechildren co-infected with MPV and RSV (Greensill, 2003, EmergingInfectious Diseases 9:372).

RSV, which belongs to the Pneumovirus genus of the subfamilyPneumoviriniae, and MPV, which belongs to the Metapneumovirus genus ofthe subfamily Pneumoviriniae, have some similarities in their geneticstructure, though MPV lacks the non-structural genes NS1 and NS2 foundin RSV. The RSV and MPV envelopes contain three virally encodedtransmembrane surface glycoproteins: the major attachment protein G, thefusion protein F, and the small hydrophobic SH protein. Although the RSVand MPV envelopes contain proteins that are functionally similar, it isimportant to note, however, that the F proteins of RSV and MPV shareonly 33% amino acid sequence identity. Further, antisera generatedagainst either RSV or MPV do not cross-neutralize both viruses (Wyde etal., 2003, Antiviral Research 60:51-59) and so far no monoclonalantibodies have been isolated that are able to cross-neutralize both RSVand MPV.

The RSV and MPV F glycoproteins direct viral penetration by fusionbetween the virion envelope and the host cell plasma membrane. Later ininfection, F protein expressed on the cell surface can mediate fusionwith neighboring cells to form syncytia (Collins et al., 1984 PNAS81:7683-7687). In both cases, the N-terminus of the F subunit that iscreated by proteolytic cleavage and contains hydrophobic stretch ofamino acids, called the fusion peptide, inserts directly into the targetmembrane to initiate fusion. After binding to the target cell andsubsequent activation, the metastable pre-fusion F protein undergoes aseries of structural rearrangements that result in the insertion of thefusion peptide into the target cell membrane, followed by the formationof a stable helical bundle that forms as the viral and cell membranesare apposed. These structural changes lead to the formation of a stablepost-fusion F protein.

Vaccines for RSV or MPV infection are currently not available. Aformalin-inactivated and alum-adjuvanted RSV vaccine (FI-RSV) tested inthe 1960s was found to predispose infants for enhanced disease followingnatural RSV infection leading to high fever and severe pneumonia,resulting in high hospitalization rates and even some fatalities(Fulginiti et al., 1969, American Journal of Epidemiology 89:435-448;Kapikian et al., 1969, American Journal of Epidemiology 89:405-421; Kimet al., 1969, American Journal of Epidemiology 89:422-434). Similarly,formalin-inactivated MPV vaccines showed immune-mediated enhanceddisease in young cynomolgus macaques (de Swart et al., 2007, Vaccine25:8518-8528). Further, antiviral therapies such as Ribavirin have notbeen proven to be effective in RSV or MPV infection.

Evidence for the role of serum antibodies in protection against RSVvirus has emerged from epidemiological as well as animal studies. Ininfants, titers of maternally transmitted antibodies correlate withresistance to serious disease (Glezen et al., 1981, The Journal ofPediatrics 98:708-715) and in adults incidence and severity of lowerrespiratory tract involvement is diminished in the presence of highlevels of serum RSV neutralizing antibodies (McIntosh et al., 1978, TheJournal of Infectious Diseases 138:24-32). A monoclonal antibody,Palivizumab (Synagis), is registered for the prevention of RSV infectionin premature newborns. Palivizumab, however, is not always effective inpreventing RSV infection and is not effective therapeutically. Further,prolonged pulmonary replication of RSV in the presence of Palivizumab isfollowed in animals by the appearance of resistant virus strains (Zhaoand Sullender, 2005, Journal of Virology 79:3962-3968). Currently thereare no monoclonal antibodies for the treatment or prevention of MPVinfection.

The lack of a good working animal model for the most severe forms of RSVinfection is related to the fact that RSV and MPV are host-restrictedPneumovirus pathogens. The development of new drugs for the therapy ofRSV and MPV infections has been hampered by the lack of an animal modelable to recapitulate all the symptoms and severity of the human disease.Indeed, RSV and MPV are not a natural mouse pathogen and induce only alimited, minimally symptomatic, and rapidly aborted primary infection inresponse to a massive, non-physiologic inoculum of the virus. Pneumoniavirus of mice (PVM) is a natural rodent Pneumovirus pathogen whichbelongs to the same family, subfamily and genus (Pneumovirus) of humanand bovine RSV. The PVM F protein shares only 40% amino acid identitywith human RSV F protein, but has the same genetic organization with theexception of the M2-L overlap which is present in RSV but absent in PVM.The infection by the natural mouse pathogen PVM replicates many of thesigns and symptoms of the most severe forms of RSV as it occurs in humaninfants. PVM infection is characterized by rapid virus replicationaccompanied by a massive inflammatory response that leads to respiratoryfailure and death (Rosemberg and Domachowske, 2008, Immunology Letter118:6-12). PVM infection in mice is therefore considered to be the mostrelevant animal model of RSV and MPV severe infections of humans.

The lack of preventive treatment for MPV infection and of vaccinesagainst RSV and MPV infections as well as the therapeutic inefficacy ofPalivizumab, highlight the need for new preventive and therapeuticagents against these prominent human pathogens. Given the largeprevalence and the possibility of co-infection, it would be highlydesirable to have a single agent that is capable of preventing as wellas treating or attenuating both RSV and MPV infection and to have ananimal model in which to test the agent. Therefore, there is a need forbroadly cross-reactive neutralising antibodies that protect against awide range of paramyxoviruses, for example, at least RSV and MPV, andpreferably RSV, MPV and PVM.

SUMMARY

The invention is based, in part, on the discovery of broadlyneutralizing antibodies that neutralize infection of RSV, MPV, and PVM,as well as polypeptides to which the antibodies of the invention bind.Accordingly, in one aspect of the invention, the invention comprises anisolated antibody, for example a monoclonal antibody, a human antibody,a human monoclonal antibody, an antibody variant, or an antigen bindingfragment, that cross-neutralizes infection of RSV, MPV, and PVM.

In one embodiment of the invention, the invention comprises an isolatedantibody, or an antigen binding fragment thereof, that neutralizesinfection of both RSV and MPV. In another embodiment of the invention,the invention comprises an antibody, or an antigen binding fragmentthereof, that neutralizes infection of RSV, MPV, and PVM.

In another embodiment of the invention, the invention comprises anisolated antibody, or an antigen binding fragment thereof, thatspecifically binds RSV pre-fusion F protein and not RSV post-fusion Fprotein. In yet another embodiment, the invention comprises an isolatedantibody, or an antigen binding fragment thereof, that specificallybinds the pre-fusion F protein and not the post-fusion F protein of RSVand MPV. In yet another embodiment, the invention comprises an isolatedantibody, or an antigen binding fragment thereof, that specificallybinds the pre-fusion F protein and not the post-fusion F protein of RSV,MPV and PVM.

In another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, comprising at leastone complementarity determining region (CDR) sequence having at least95% sequence identity to any one of SEQ ID NOs: 1-6, 19-23, 35, 39-42,or 53-54 wherein the antibody neutralizes infection of RSV, MPV, andPVM.

In another embodiment of the invention, the invention comprises anantibody or antigen binding fragment thereof, comprising a heavy chainCDR1 with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 19; aheavy chain CDR2 with the amino acid sequence of SEQ ID NO: 2, SEQ IDNO: 20 or SEQ ID NO: 39; and a heavy chain CDR3 with the amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 21, SEQ ID NO: 40 or SEQ ID NO: 53,wherein the antibody neutralizes infection of RSV, MPV, and PVM. Inanother embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, comprising a lightchain CDR1 with the amino acid sequence of SEQ ID NO: 4; a light chainCDR2 with the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 22 or SEQID NO: 41; and a light chain CDR3 with the amino acid sequence of SEQ IDNO: 6, SEQ ID NO: 23, SEQ ID NO: 35, SEQ ID NO: 42 or SEQ ID NO: 54,wherein the antibody neutralizes infection of RSV, MPV, and PVM.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, wherein the antibodycomprises: (i) the heavy chain CDR1, CDR2, and CDR3 sequences as setforth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, andthe light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO: 35, respectively; (ii) the heavychain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQID NO: 2, and SEQ ID NO: 3, respectively, and the light chain CDR1,CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, andSEQ ID NO: 6, respectively; (iii) the heavy chain CDR1, CDR2, and CDR3sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 39, and SEQ ID NO:40, respectively, and the light chain CDR1, CDR2, and CDR3 sequences asset forth in SEQ ID NO: 4, SEQ ID NO: 41, and SEQ ID NO: 42,respectively (iv) the heavy chain CDR1, CDR2, and CDR3 sequences as setforth in SEQ ID NO: 1, SEQ ID NO: 39, and SEQ ID NO: 40, respectively,and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; (v) the heavychain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQID NO: 2, and SEQ ID NO: 3, respectively, and the light chain CDR1,CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 41,and SEQ ID NO: 42, respectively, and wherein the antibody neutralizesinfection of RSV, MPV, and PVM.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, wherein the antibodycomprises: (i) the heavy chain CDR1, CDR2, and CDR3 sequences as setforth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively,and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQID NO: 4, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; (ii) the heavychain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQID NO: 39, and SEQ ID NO: 53, respectively, and the light chain CDR1,CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 41,and SEQ ID NO: 54, respectively; (iii) the heavy chain CDR1, CDR2, andCDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 39, and SEQ IDNO: 53, respectively, and the light chain CDR1, CDR2, and CDR3 sequencesas set forth in SEQ ID NO: 4, SEQ ID NO: 22, and SEQ ID NO: 23,respectively; or (iv) the heavy chain CDR1, CDR2, and CDR3 sequences asset forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21,respectively, and the light chain CDR1, CDR2, and CDR3 sequences as setforth in SEQ ID NO: 4, SEQ ID NO: 41, and SEQ ID NO: 54, respectively,and wherein the antibody neutralizes infection of RSV, MPV, and PVM.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, wherein the antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 17 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 37; or a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 13 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:14; or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 17 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 14; or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 49 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 50; ora heavy chain variable region comprising the amino acid sequence of SEQID NO: 49 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 14; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 17 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 50, and whereinthe antibody neutralizes infection of RSV, MPV, and PVM.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, wherein the antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 29 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 30; or a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 33 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:30 or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 59 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 60; or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 59 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 30; ora heavy chain variable region comprising the amino acid sequence of SEQID NO: 33 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 60, and wherein the antibody neutralizesinfection of RSV, MPV, and PVM.

The invention further comprises an antibody, or an antigen bindingfragment thereof, described herein as HMB3210 (or 3210); or HMB2430 (or2430). In another embodiment, the invention comprises an antibody, orantigen binding fragment thereof, that neutralizes infection of RSV,MPV, and PVM, wherein the antibody or fragment thereof is expressed byan immortalized B cell clone that produces HMB3210 or HMB2430.

In another aspect, the invention comprises a nucleic acid moleculecomprising a polynucleotide encoding an antibody or antibody fragment ofthe invention. In yet another aspect, the invention comprises a vectorcomprising a nucleic acid molecule of the invention. The invention alsocomprises a cell that expresses an antibody of the invention or anantigen binding fragment thereof. In still another aspect, the inventioncomprises an isolated or purified immunogenic polypeptide comprising anepitope that binds to an antibody or antigen binding fragment of theinvention.

The invention further comprises a pharmaceutical composition comprisingan antibody of the invention or an antigen binding fragment thereof, anucleic acid molecule of the invention, a vector comprising a nucleicacid molecule of the invention, a cell expressing an antibody or anantibody fragment of the invention, or an immunogenic polypeptide of theinvention, and a pharmaceutically acceptable diluent or carrier. Theinvention also comprises a pharmaceutical composition comprising a firstantibody or an antigen binding fragment thereof, and a second antibody,or an antigen binding fragment thereof, wherein the first antibody is anantibody of the invention, and the second antibody is an antibody, or anantigen binding fragment thereof, that neutralizes infection of RSV orMPV or both RSV and MPV, or all three of RSV, MPV, and PVM.

Use of an antibody of the invention, or an antigen binding fragmentthereof, a nucleic acid of the invention, a vector comprising a nucleicacid of the invention, a cell expressing a vector of the invention, anisolated or purified immunogenic polypeptide comprising an epitope thatbinds to an antibody or antibody fragment of the invention, or apharmaceutical composition of the invention (i) in the manufacture of amedicament for the treatment or attenuation of RSV or MPV or both RSVand MPV co-infection, (ii) in a vaccine, or (iii) in diagnosis of RSVand/or MPV virus infection is also contemplated to be within the scopeof the invention. Further, use of an antibody of the invention, or anantigen binding fragment thereof, for monitoring the quality of avaccine against RSV or MPV or both RSV and MPV by checking that theantigen of said vaccine contains the specific epitope in the correctconformation is also contemplated to be within the scope of theinvention.

In another aspect, the invention comprises a method of treating orattenuating RSV and MPV infection or lowering the risk of RSV and MPVinfection comprising administering to a subject in need thereof, atherapeutically effective amount of an antibody or an antigen bindingantibody fragment of the invention.

In a further aspect, the invention comprises a polypeptide whichspecifically binds to an antibody of the invention, or an antigenbinding fragment thereof, for use (i) in therapy, (ii) in themanufacture of a medicament for the treatment or attenuation of RSV orMPV or both RSV and MPV infection, (iii) as a vaccine, or (iv) inscreening for ligands able to neutralise infection of RSV or MPV or bothRSV and MPV.

DESCRIPTION OF FIGURES

FIG. 1 shows the results of the screening of monoclonal antibodiesproduced by EBV-immortalized memory B cells from 7 donors (Don. 1 to 7)for their ability to neutralize RSV or MPV virus infection in vitro.

FIG. 2 shows the results of neutralization of RSV and MPV by monoclonalantibodies HMB2430, HMB3210, 234 mAb and Palivizumab.

FIG. 3 shows the binding to RSV F or Tetanus Toxin protein by monoclonalantibodies Motavizumab, 234 mAb, HMB2430 and HMB3210 as measured byELISA.

FIG. 4 shows the binding of labeled monoclonal antibodies toRSV-infected Hep-2 cells in the presence of large excess of theindicated unlabeled antibodies.

FIG. 5 shows the binding of monoclonal antibodies HMB2430, HMB3210, 234mAb, and Motavizumab to RSV F protein from lysates of RSV-Hep-2-infectedcells under reducing or non-reducing conditions as measured by Westernblot analysis.

FIG. 6 shows the binding of monoclonal antibodies HMB3210 and 234 mAb toMPV F protein from lysates of MPV-LLC-MK2-infected cells under reducingor non-reducing conditions as measured by Western blot analysis.

FIG. 7 shows the results of neutralization of RSV Long strain and thePZ-MARM6 isolate by monoclonal antibodies HMB2430, HMB3210, 234 mAb andPalivizumab.

FIG. 8 shows the results of the analysis of HMB3210v2 on a reducingSDS-PAGE gel following incubation in the presence (+) or absence (−) ofthe N-glycosidase PNG-ase F. Highlighted with the black box is the minorfraction of the HMB3210 light chain which is glycosylated.

FIG. 9 shows the results of neutralization of MPV I-PV 03/01-6621 andRSV A2 by monoclonal antibodies HMB3210v2 and HMB3210v3.

FIG. 10 shows the results of neutralization of a panel of MPV and RSVstrains by monoclonal antibodies HMB3210v2 and HMB3210v3.

FIG. 11 shows the results of neutralization of MPV I-PV 03/01-6621 andRSV A2 by HMB3210 and HMB2430 monoclonal antibody germlined variants.

FIG. 12 shows the results of size exclusion chromatography analysis ofthe RSV F post-fusion recombinant protein co-incubated or not withHMB3210 or Palivizumab.

FIG. 13 shows the results of size exclusion chromatography analysis ofthe RSV F pre-fusion recombinant protein co-incubated or not withHMB3210 or Palivizumab.

FIG. 14 shows the binding of HMB3210v3 or Palivizumab (PVZ) to thepre-fusion and post-fusion RSV F proteins as measured by surface plasmonresonance (SPR).

FIG. 15 shows the virus neutralization and inhibition of viral spreadingby human monoclonal antibodies HMB3210v3 and D25.

FIGS. 16A and 16B show the prophylactic efficacy of HMB3210v3 andPalivizumab in RSV or MPV infection.

FIG. 17 shows the therapeutic efficacy of HMB3210 in STAT1 deficientmice infected with RSV.

FIGS. 18A-18D show the prophylactic and therapeutic efficacy of HMB3210in mice infected with a lethal dose of PVM.

FIG. 19 shows the blocking in the increase of lung viral titers in micetreated with HMB3210v3 on day 3, 4 or 5 after lethal infection with PVM.

FIGS. 20A and 20B show the prophylactic and therapeutic efficacy ofHMB3210v3 variants bearing the wild type Fc or the LALA mutation in miceinfected with a lethal dose of PVM.

FIG. 21 shows an alignment highlighting the high conservation of theYLSALR peptide recognized by HMB3210 in RSV, BRSV, PVM and MPV sequencesas compared to parainfluenza virus 5 (PIVS).

FIG. 22 shows a model of the pre-fusion RSV F protein showing thelocation of the YLSALR peptide and of the neighboring Palivizumab (PVZ)site and ribbon diagrams highlighting the rearrangement of the PVZ andHMB3210v3 sites in the pre- and post-fusion conformation of the RSV Fprotein.

FIG. 23 shows the high degree of conservation of the HMB3210 coreepitope in 364 RSV, 162 MPV, 8 BRSV and 5 PVM strains.

FIGS. 24A, 24B and 24C show the sequences for the various heavy andlight chain variants for HMB3210.

FIGS. 25A and 25B show the sequences for the various heavy and lightchain variants for HMB2430.

DETAILED DESCRIPTION

The invention is based, in part, on the discovery and isolation ofantibodies that cross-neutralize both RSV and MPV or RSV, MPV, and PVM,as well as epitopes to which the antibodies of the invention bind. Suchantibodies are desirable, as only one or few antibodies are required inorder to neutralize both RSV and MPV or RSV, MPV, and PVM. Further, thecross-neutralizing antibodies are produced at high titers to reducecosts of production of medicaments comprising the antibodies for thetreatment of RSV and/or MPV infection. In addition, the epitopesrecognized by such antibodies may be part of a vaccine capable ofinducing broad protection against both RSV and MPV.

Although the antibodies of the invention neutralize RSV, MPV, and PVM,in some embodiments of the invention, for example those related to thetreatment of disease, development of vaccines, etc., the currentdisclosure refers only to RSV and MPV, as these viruses are humanpathogens, while PVM is a mouse pathogen. As used herein, the terms“both RSV and MPV,” and “RSV, MPV and PVM” are used interchangeablybased on the context.

Accordingly, in one aspect, the invention provides an isolated antibody,antibody variants and antigen binding fragments thereof, that neutralizeboth RSV and MPV or RSV, MPV, and PVM. In one embodiment, the RSV is ahuman RSV. In another embodiment, the RSV is a bovine RSV. Theantibodies of the invention neutralize both human RSV (hRSV) and bovineRSV (bRSV). In another embodiment, the MPV is a human MPV.

In one embodiment, the invention also provides an isolated antibody, oran antigen binding fragment thereof, that neutralizes infection of bothgroup A and group B RSV. In another embodiment, the invention providesan isolated antibody, or an antigen binding fragment thereof, thatneutralizes infection of both group A and group B MPV. In yet anotherembodiment, the invention provides an isolated antibody, or an antigenbinding fragment thereof, that neutralizes infection of both group A andgroup B RSV as well as both group A and group B MPV.

As discussed earlier, RSV, MPV, and PVM have some similarities in theirgenetic structure. The amino acid sequences of the G and F proteins areclassified into A and B groups in both RSV and MPV; MPV is furtherdivided in 4 subgroups: A1, A2, B1 and B2. PVM is not subdivided intogroups or sub-groups. The RSV, MPV or PVM F protein is a type Itransmembrane surface protein that has an N-terminal cleaved signalpeptide and a membrane anchor near the C-terminus. RSV and MPV Fproteins are synthesized as inactive FO precursors that assemble intohomotrimers and are activated by cleavage. The F protein is formed bythree domains (DI to DIII), a fusion peptide (FP) and threeheptad-repeats regions (HR-A, -B and -C). The RSV and MPV Fglycoproteins direct viral penetration by fusion between the virionenvelope and the host cell plasma membrane. In both cases, theN-terminus of the F subunit, that is created by proteolytic cleavage andcontains the fusion peptide, inserts directly into the target membraneto initiate fusion. After binding to the target cell and subsequentactivation, the metastable pre-fusion F protein undergoes a series ofstructural rearrangements that result in the insertion of the fusionpeptide into the target cell membrane, followed by the formation of astable helical bundle that forms as the viral and cell membranes areapposed. These structural changes lead to the formation of a stablepost-fusion F protein. Later in infection, the F protein expressed onthe cell surface of infected cells can mediate fusion with adjacentnon-infected cells forming large syncytia.

The epitopes for Palivizumab and Motavizumab have been mapped on thepost-fusion RSV F protein antigenic site II (also called site A) formedby residues 255-275. MAB19 and 101F target the post-fusion RSV F proteinantigenic site IV (also called site C) of RSV formed by residues422-438. MAB19 was tested in clinical trials but failed to showsignificant efficacy (Johnson et al., 1999, The Journal of InfectiousDiseases 180:35-40; Meissner et al., 1999, Antimicrobial Agents andChemotherapy 43:1183-1188).

To be effective, antibodies should recognize the pre-fusion F protein,which is the relevant conformation to block virus entry, and preferablyavoids recognition of the abundant post-fusion F protein that can act asa decoy, thus consuming the antibody and reducing its efficacy. So farno antibodies recognizing the RSV pre-fusion but not the RSV post-fusionF protein have been isolated.

In one embodiment of the invention, the invention comprises an isolatedantibody, or an antigen binding fragment thereof, that specificallybinds RSV pre-fusion F protein and not RSV post-fusion F protein. Inanother embodiment, the invention comprises an isolated antibody, or anantigen binding fragment thereof, that specifically binds the pre-fusionF protein and not the post-fusion F protein of RSV and MPV. In anotherembodiment, the invention provides antibodies that specifically bind tothe pre-fusion F protein but not to the post-fusion F protein of RSV,MPV and PVM.

The invention provides antibodies that bind to the F protein of RSV, MPVand PVM. Despite the fact that there is only approximately 33% and 40%amino acid sequence identity between RSV and MPV or RSV and PVM Fproteins, respectively, the antibodies of the invention recognize ashared epitope present on RSV, MPV and PVM F proteins. This epitope isdifferent from all those recognized by the hitherto know antibodies suchas Palivizumab, Motavizumab, mAb 101F etc. The antibodies of theinvention do not, for example, bind the antigenic site II (recognized byMotavizumab and Palivizumab), nor the antigenic site IV (recognized bymAb 101F), nor the antigenic site I (bound by mAb 131-2A). The epitopesrecognized by the antibodies of the invention on the RSV F protein arealso distinct from that recognized by the mAb D25, an antibody specificonly to RSV. In addition, the epitopes recognized by the antibodies ofthe invention on the MPV F protein are distinct from that recognized bythe mAb 234 (that recognizes an epitope on the MPV F protein whichcorrespond to the antigenic site II on RSV F protein). In general, theantibodies of the invention recognize a conformational epitope. In oneembodiment, the conformational epitope is present only undernon-reducing conditions. In another embodiment, the conformationalepitope relies on the presence of disulphide bonds between amino acidresidues on the F protein.

As shown herein, the antibodies or antigen binding fragments of theinvention bind specifically to several different strains of both RSV andMPV and neutralize both RSV and MPV. Further, the antibodies or antigenbinding fragments of the invention bind specifically to, andcross-neutralize both group A and group B RSV as well as both group Aand group B MPV, including all corresponding MPV subgroups (i.e. A1, A2,B1, and B2).

The antibody and antigen binding fragment of the invention have highneutralizing potency. The concentration of the antibody of the inventionrequired for 50% neutralization of RSV, MPV and PVM, is, for example,about 500 ng/ml or less. In one embodiment, the concentration of theantibody of the invention required for 50% neutralization of RSV, MPVand PVM is about 500, 450, 400, 350, 300, 250, 200, 175, 150, 125, 100,90, 80, 70, 60 or about 50 ng/ml or less. This means that only lowconcentrations of antibody are required for 50% neutralization of RSV,MPV and PVM. Specificity and potency can be measured using standardassays as known to one of skill in the art.

The antibodies of the invention may be human antibodies, monoclonalantibodies, human monoclonal antibodies, recombinant antibodies orpurified antibodies. The invention also provides fragments of theantibodies of the invention, particularly fragments that retain theantigen-binding activity of the antibodies. Such fragments include, butare not limited to, single chain antibodies, Fab, Fab′, F(ab′)2, Fv orscFv. Although the specification, including the claims, may, in someplaces, refer explicitly to antigen binding fragment(s), antibodyfragment(s), variant(s) and/or derivative(s) of antibodies, it isunderstood that the term “antibody” or “antibody of the invention”includes all categories of antibodies, namely, antigen bindingfragment(s), antibody fragment(s), variant(s) and derivative(s) ofantibodies.

The sequences of the heavy chains and light chains of several antibodiesof the invention, each comprising three CDRs on the heavy chain andthree CDRs on the light chain have been determined. The position of theCDR amino acids are defined according to the IMGT numbering system. Thesequences of the CDRs, heavy chains, light chains as well as thesequences of the nucleic acid molecules encoding the CDRs, heavy chains,light chains of the antibodies of the invention are disclosed in thesequence listing. The CDRs of the antibody heavy chains are referred toas CDRH1 (or HCDR1), CDRH2 (or HCDR2) and CDRH3 (or HCDR3),respectively. Similarly, the CDRs of the antibody light chains arereferred to as CDRL1 (or LCDR1), CDRL2 (or LCDR2) and CDRL3 (or LCDR3),respectively. Table 1 provides the SEQ ID numbers for the amino acidsequences of the six CDRs of the heavy and light chains, respectively,of the exemplary antibodies of the invention.

TABLE 1 SEQ ID Numbers for CDR Polypeptides of Antibodies thatNeutralize RSV, MPV and PVM. SEQ ID NOs. for CDR Polypeptides CDRH1CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 3210 variant 1 1 2 3 4 5 6 3210 variant 21 2 3 4 5 6 3210 variant 3 1 2 3 4 5 35 3210 variant 4 1 39 40 4 41 423210 variant 5 1 39 40 4 5 6 3210 variant 6 1 2 3 4 41 42 2430 variant 119 20 21 4 22 23 2430 variant 2 19 20 21 4 22 23 2430 variant 3 1 39 534 41 54 2430 variant 4 1 39 53 4 22 23 2430 variant 5 19 20 21 4 41 54

In one embodiment, an antibody or antibody fragment of the inventioncomprises at least one CDR with a sequence that has at least 95%sequence identity to any one of SEQ ID NOs: 1-6, 19-23, 35, 39-42, or53-54. The CDRs of the variants of the antibody 3210 and antibody 2430are provided in FIGS. 24 and 25 respectively (CDRs are highlighted inbold).

In another embodiment, the invention provides an antibody or antigenbinding fragment comprising a heavy chain comprising one or more (i.e.one, two or all three) heavy chain CDRs from 3210 variant 1, 3210variant 2, 3210 variant 3, 3210 variant 4, 3210 variant 5, 3210 variant6, 2430 variant 1, 2430 variant 2, 2430 variant 3, 2430 variant 4 or2430 variant 5.

In yet another embodiment, the antibody or antigen binding fragment ofthe invention comprises a heavy chain CDR1 with the amino acid sequenceof SEQ ID NO: 1 or SEQ ID NO: 19; a heavy chain CDR2 with the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 20 or SEQ ID NO: 39; and a heavychain CDR3 with the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 21,SEQ ID NO: 40 or SEQ ID NO: 53. In certain embodiments, an antibody orantibody fragment as provided herein comprises a heavy chain comprisingthe amino acid sequence of (i) SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 forCDRH2 and SEQ ID NO: 3 for CDRH3, (ii) SEQ ID NO: 1 for CDRH1, SEQ IDNO: 39 for CDRH2, and SEQ ID NO: 40 for CDRH3, (iii) SEQ ID NO: 19 forCDRH1, SEQ ID NO; 20 for CDRH2, and SEQ ID NO: 21 for CDRH3, or (iv) orSEQ ID NO: 1 for CDRH1, SEQ ID NO: 39 for CDRH2, and SEQ ID NO: 53 forCDRH3.

Also provided is an antibody or antigen binding fragment comprising alight chain comprising one or more (i.e. one, two or all three) lightchain CDRs from 3210 variant 1, 3210 variant 2, 3210 variant 3, 3210variant 4, 3210 variant 5, 3210 variant 6, 2430 variant 1, 2430 variant2, 2430 variant 3, 2430 variant 4 or 2430 variant 5. In one embodiment,the antibody or antigen binding fragment of the invention comprises alight chain CDR1 with the amino acid sequence of SEQ ID NO: 4; a lightchain CDR2 with the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 22or SEQ ID NO: 41; and a light chain CDR3 with the amino acid sequence ofSEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 35, SEQ ID NO: 42 or SEQ ID NO:54. In certain embodiments, an antibody or antibody fragment as providedherein comprises a light chain comprising the amino acid sequence of (i)SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 for CDRL2, and SEQ ID NO: 6 forCDRL3; (ii) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 for CDRL2, and SEQ IDNO: 35 for CDRL3; (iii) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 41 for CDRL2,and SEQ ID NO: 42 for CDRL3; (iv) SEQ ID NO: 4 for CDRL1, SEQ ID NO; 22for CDRL2, and SEQ ID NO: 23 for CDRL3; or (v) SEQ ID NO: 4 for CDRL1,SEQ ID NO: 41 for CDRL2, and SEQ ID NO: 54 for CDRL3.

In one embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 3210 variant 1as listed in Table 1, and neutralizes infection of RSV, MPV and PVM. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 3210 variant 2as listed in Table 1, and neutralizes infection of RSV, MPV and PVM. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 3210 variant 3as listed in Table 1, and neutralizes infection of RSV, MPV and PVM. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 3210 variant 4as listed in Table 1, and neutralizes infection of both RSV, MPV andPVM. In another embodiment, an antibody of the invention, or antigenbinding fragment thereof, comprises all of the CDRs of antibody 3210variant 5 as listed in Table 1, and neutralizes infection of both RSV,MPV and PVM. In another embodiment, an antibody of the invention, orantigen binding fragment thereof, comprises all of the CDRs of antibody3210 variant 6 as listed in Table 1, and neutralizes infection of bothRSV, MPV and PVM.

In yet another embodiment, an antibody of the invention, or antigenbinding fragment thereof, comprises all of the CDRs of antibody 2430variant 1 as listed in Table 1, and neutralizes infection of both RSV,MPV and PVM. In another embodiment, an antibody of the invention, orantigen binding fragment thereof, comprises all of the CDRs of antibody2430 variant 2 as listed in Table 1, and neutralizes infection of bothRSV, MPV and PVM. In another embodiment, an antibody of the invention,or antigen binding fragment thereof, comprises all of the CDRs ofantibody 2430 variant 3 as listed in Table 1, and neutralizes infectionof both RSV, MPV and PVM. In another embodiment, an antibody of theinvention, or antigen binding fragment thereof, comprises all of theCDRs of antibody 2430 variant 4 as listed in Table 1, and neutralizesinfection of both RSV, MPV and PVM. In another embodiment, an antibodyof the invention, or antigen binding fragment thereof, comprises all ofthe CDRs of antibody 2430 variant 5 as listed in Table 1, andneutralizes infection of both RSV, MPV and PVM.

The SEQ ID numbers for the amino acid sequence for the heavy chainvariable region (VH) and the light chain variable region (VL) ofexemplary antibodies of the invention as well as the SEQ ID numbers forthe nucleic acid sequences encoding them are listed in Table 2.

TABLE 2 SEQ ID Numbers for V_(H) and V_(L) amino acid and nucleic acidresidues for Antibodies that Neutralize RSV, MPV and PVM. SEQ ID NOs.for V_(H) and V_(L) amino acid and nucleic acid residues V_(H) V_(L)V_(H) V_(L) V_(H) V_(L) amino amino nucleic nucleic chain chain acidacid acid acid 3210 variant 1 VH.1 VL 13 14 15 16 3210 variant 2 VH.2 VL17 14 18 16 3210 variant 3 VH.2 VL.3 17 37 18 38 3210 variant 4 VH.3VL.4 49 50 51 52 3210 variant 5 VH.3 VL 49 14 51 16 3210 variant 6 VH.2VL.4 17 50 18 52 2430 variant 1 VH.1 VL 29 30 31 32 2430 variant 2 VH.2VL 33 30 34 32 2430 variant 3 VH.3 VL.2 59 60 61 62 2430 variant 4 VH.3VL 59 30 61 32 2430 variant 5 VH.2 VL.2 33 60 34 62

In one embodiment, an antibody or antibody fragment of the inventioncomprises a heavy chain variable region having an amino acid sequencethat is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the sequence recited in any one of SEQ ID NOs: 13, 17, 29,33, 49 or 59. In another embodiment, an antibody or antibody fragment ofthe invention comprises a light chain variable region having an aminoacid sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to the sequence recited in SEQ ID NOs: 14, 30, 37, 50or 60. In yet another embodiment, an antibody or antibody fragment ofthe invention comprises a heavy chain or a light chain variable regionhaving an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to the sequences provided in FIGS.24 and 25.

In another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, that neutralizesinfection of RSV, MPV and PVM and comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 13 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:14; or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 13 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 37; or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 13 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 50; ora heavy chain variable region comprising the amino acid sequence of SEQID NO: 17 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 14; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 17 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 37; or a heavychain variable region comprising the amino acid sequence of SEQ ID NO:49 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 50; or a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 49 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 14; or a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 49 anda light chain variable region comprising the amino acid sequence of SEQID NO: 37; or a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 17 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 50.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, that neutralizesinfection of RSV, MIN and PVM and comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 29 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:30; or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 29 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 60; or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 33 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 30; ora heavy chain variable region comprising the amino acid sequence of SEQID NO: 59 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 60; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 59 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 30; or a heavychain variable region comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 60.

Examples of antibodies of the invention include, but are not limited to,HMB3210 variant 1, HMB3210 variant 2, HMB3210 variant 3, HMB3210 variant4, HMB3210 variant 5, HMB3210 variant 6, HMB2430 variant 1, HMB2430variant 2, HMB2430 variant 3, HMB2430 variant 4 or HMB2430 variant 5.

The invention further comprises an antibody, or fragment thereof, thatbinds to the same epitope as an antibody or antigen binding fragment ofthe invention, or an antibody that competes with an antibody or antigenbinding fragment of the invention.

As can be seen from Tables 1 and 2, the CDRs, heavy chains and lightchains of the disclosed antibodies can be interchanged to provide newantibodies that retain their binding and neutralizing capabilities.Antibodies of the invention thus include antibodies and antigen bindingfragments comprising any combination of the CDRs provided in Table 1 orheavy and light chains provided in Table 2.

Antibodies of the invention also include hybrid antibody molecules thatcomprise one or more CDRs from an antibody of the invention and one ormore CDRs from another antibody to the same epitope. In one embodiment,such hybrid antibodies comprise three CDRs from an antibody of theinvention and three CDRs from another antibody to the same epitope.Exemplary hybrid antibodies comprise (i) the three light chain CDRs froman antibody of the invention and the three heavy chain CDRs from anotherantibody to the same epitope, or (ii) the three heavy chain CDRs from anantibody of the invention and the three light chain CDRs from anotherantibody to the same epitope.

Variant antibodies are also included within the scope of the invention.Thus, variants of the sequences recited in the application are alsoincluded within the scope of the invention. Such variants includenatural variants generated by somatic mutation in vivo during the immuneresponse or in vitro upon culture of immortalized B cell clones.Alternatively, variants may arise due to the degeneracy of the geneticcode or may be produced due to errors in transcription or translation.

Further variants of the antibody sequences having improved affinityand/or potency may be obtained using methods known in the art and areincluded within the scope of the invention. For example, amino acidsubstitutions may be used to obtain antibodies with further improvedaffinity. Alternatively, codon optimization of the nucleotide sequencemay be used to improve the efficiency of translation in expressionsystems for the production of the antibody. Further, polynucleotidescomprising a sequence optimized for antibody specificity or neutralizingactivity by the application of a directed evolution method to any of thenucleic acid sequences of the invention are also within the scope of theinvention.

In one embodiment variant antibody sequences may share 70% or more (i.e.75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) amino acid sequenceidentity with the sequences recited in the application. In someembodiments such sequence identity is calculated with regard to the fulllength of the reference sequence (i.e. the sequence recited in theapplication). In some further embodiments, percentage identity, asreferred to herein, is as determined using BLAST version 2.1.3 using thedefault parameters specified by the NCBI (the National Center forBiotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62matrix; gap open penalty=11 and gap extension penalty=1].

In another aspect, the invention also includes nucleic acid sequencesencoding part or all of the light and heavy chains and CDRs of theantibodies of the present invention. Provided herein are nucleic acidsequences encoding part or all of the light and heavy chains and CDRs ofexemplary antibodies of the invention. Table 2 provides the SEQ IDnumbers for the nucleic acid sequences encoding the heavy chain andlight chain variable regions of some examples of antibodies of theinvention. Table 3 provides the SEQ ID numbers for the nucleic acidsequences encoding the CDRs of the exemplary antibodies of theinvention. Due to the redundancy of the genetic code, variants of thesenucleic acid sequences will exist that encode the same amino acidsequences.

TABLE 3 SEQ ID Numbers for CDR Polynucleotides of Antibodies thatNeutralize RSV, MPV and PVM. SEQ ID NOs. for CDR Polynucleotides CDRH1CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 3210 variant 1 7 8 9 10 11 12 3210 variant2 7 8 9 10 11 12 3210 variant 3 7 8 9 10 11 36 3210 variant 4 43 44 4546 47 48 3210 variant 5 43 44 45 10 11 12 3210 variant 6 7 8 9 46 47 482430 variant 1 24 25 26 10 27 28 2430 variant 2 24 25 26 10 27 28 2430variant 3 55 44 56 57 47 58 2430 variant 4 55 44 56 10 27 28 2430variant 5 24 25 26 57 47 58

In one embodiment, nucleic acid sequences according to the inventioninclude nucleic acid sequences having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to the nucleic acid encoding a heavy or light chainof an antibody of the invention. In another embodiment, a nucleic acidsequence of the invention has the sequence of a nucleic acid encoding aheavy or light chain CDR of an antibody of the invention. For example, anucleic acid sequence according to the invention comprises a sequencethat is at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% identical to the nucleic acidsequences of SEQ ID NOs: 7-12, 15, 16, 18, 24-28, 31-32, 34, 36, 38,43-48, 51-52, 55-58, or 61-62.

In yet another embodiment, nucleic acid sequences according to theinvention include nucleic acid sequences having at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identity to the nucleic acid encoding a heavy orlight chain of an antibody of the invention as provided in FIGS. 24 and25.

Further included within the scope of the invention are vectors, forexample, expression vectors, comprising a nucleic acid sequenceaccording to the invention. Cells transformed with such vectors are alsoincluded within the scope of the invention. Examples of such cellsinclude but are not limited to, eukaryotic cells, e.g., yeast cells,animal cells or plant cells. In one embodiment the cells are mammalian,e.g., human, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The invention also relates to monoclonal antibodies that bind to anepitope capable of binding the antibodies or antigen binding fragmentsof the invention.

Monoclonal and recombinant antibodies are particularly useful inidentification and purification of the individual polypeptides or otherantigens against which they are directed. The antibodies of theinvention have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (MA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labeled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme. The antibodies mayalso be used for the molecular identification and characterization(epitope mapping) of antigens.

Antibodies of the invention can be coupled to a drug for delivery to atreatment site or coupled to a detectable label to facilitate imaging ofa site comprising cells of interest, such as cells infected with RSV orMPV or both RSV and MPV. Methods for coupling antibodies to drugs anddetectable labels are well known in the art, as are methods for imagingusing detectable labels. Labeled antibodies may be employed in a widevariety of assays, employing a wide variety of labels. Detection of theformation of an antibody-antigen complex between an antibody of theinvention and an epitope of interest (an epitope or RSV or MPV or both)can be facilitated by attaching a detectable substance to the antibody.Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors,prosthetic group complexes, free radicals, particles, dyes, and thelike. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material is luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and examples ofsuitable radioactive material include 1251, 1311, 35S, or 3H. Suchlabeled reagents may be used in a variety of well-known assays, such asradioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescentimmunoassays, and the like. (See U.S. Pat. Nos. 3,766,162; 3,791,932;3,817,837; and 4,233,402 for example).

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, I-131, I-123, I-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody, or antibody fragment thereof, can beconjugated to a second antibody, or antibody fragment thereof, to forman antibody heteroconjugate as described in U.S. Pat. No. 4,676,980. Inaddition, linkers may be used between the labels and the antibodies ofthe invention (e.g., U.S. Pat. No. 4,831,175). Antibodies or,antigen-binding fragments thereof may be directly labeled withradioactive iodine, indium, yttrium, or other radioactive particle knownin the art (e.g., U.S. Pat. No. 5,595,721). Treatment may consist of acombination of treatment with conjugated and non-conjugated antibodiesadministered simultaneously or subsequently (e.g., WO00/52031;WO00/52473).

Antibodies of the invention may also be attached to a solid support.Additionally, antibodies of the invention, or functional antibodyfragments thereof, can be chemically modified by covalent conjugation toa polymer to, for example, increase their circulating half-life.Examples of polymers, and methods to attach them to peptides, are shownin U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. In someembodiments the polymers may be selected from polyoxyethylated polyolsand polyethylene glycol (PEG). PEG is soluble in water at roomtemperature and has the general formula: R(O—CH2-CH2)n O—R where R canbe hydrogen, or a protective group such as an alkyl or alkanol group. Inone embodiment the protective group may have between 1 and 8 carbons. Ina further embodiment the protective group is methyl. The symbol n is apositive integer. In one embodiment n is between 1 and 1,000. In anotherembodiment n is between 2 and 500. In one embodiment the PEG has anaverage molecular weight between 1,000 and 40,000. In a furtherembodiment the PEG has a molecular weight between 2,000 and 20,000. Inyet a further embodiment the PEG has a molecular weight between 3,000and 12,000. In one embodiment PEG has at least one hydroxy group. Inanother embodiment the PEG has a terminal hydroxy group. In yet anotherembodiment it is the terminal hydroxy group which is activated to reactwith a free amino group on the inhibitor. However, it will be understoodthat the type and amount of the reactive groups may be varied to achievea covalently conjugated PEG/antibody of the present invention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. In oneembodiment, POG is used. Without being bound by any theory, because theglycerol backbone of polyoxyethylated glycerol is the same backboneoccurring naturally in, for example, animals and humans in mono-, di-,triglycerides, this branching would not necessarily be seen as a foreignagent in the body. In some embodiments POG has a molecular weight in thesame range as PEG. Another drug delivery system that can be used forincreasing circulatory half-life is the liposome. Methods of preparingliposome delivery systems are known to one of skill in the art. Otherdrug delivery systems are known in the art and are described in, forexample, referenced in Poznansky et al. (1980) and Poznansky (1984).

Antibodies of the invention may be provided in purified form. Typically,the antibody will be present in a composition that is substantially freeof other polypeptides e.g., where less than 90% (by weight), usuallyless than 60% and more usually less than 50% of the composition is madeup of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (orheterologous) hosts e.g., in mice. In particular, the antibodies mayhave an idiotope that is immunogenic in non-human hosts, but not in ahuman host. Antibodies of the invention for human use include those thatcannot be easily isolated from hosts such as mice, goats, rabbits, rats,non-primate mammals, etc. and cannot generally be obtained byhumanization or from xeno-mice.

Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgMi.e. an α, γ or μ heavy chain), but will generally be IgG. Within theIgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass.Antibodies of the invention may have a κ or a λ light chain.

Production of Antibodies

Antibodies according to the invention can be made by any method known inthe art. For example, the general methodology for making monoclonalantibodies using hybridoma technology is well known (Kohler, G. andMilstein, C., 1975; Kozbar et al. 1983). In one embodiment, thealternative EBV immortalization method described in WO2004/076677 isused.

Using the method described in WO 2004/076677, B cells producing theantibody of the invention can be transformed with EBV and a polyclonal Bcell activator. Additional stimulants of cellular growth anddifferentiation may optionally be added during the transformation stepto further enhance the efficiency. These stimulants may be cytokinessuch as IL-2 and IL-15. In one aspect, IL-2 is added during theimmortalization step to further improve the efficiency ofimmortalization, but its use is not essential. The immortalized B cellsproduced using these methods can then be cultured using methods known inthe art and antibodies isolated therefrom.

Using the method described in WO 2010/046775, plasma cells can becultured in limited numbers, or as single plasma cells in microwellculture plates. Antibodies can be isolated from the plasma cellcultures. Further, from the plasma cell cultures, RNA can be extractedand PCR can be performed using methods known in the art. The VH and VLregions of the antibodies can be amplified by RT-PCR, sequenced andcloned into an expression vector that is then transfected into HEK293Tcells or other host cells. The cloning of nucleic acid in expressionvectors, the transfection of host cells, the culture of the transfectedhost cells and the isolation of the produced antibody can be done usingany methods known to one of skill in the art.

The antibodies may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Techniques for purification of antibodies,e.g., monoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention. For example, the invention includes a scFv comprising theCDRs from an antibody of the invention. Also included are heavy or lightchain monomers and dimers, single domain heavy chain antibodies, singledomain light chain antibodies, as well as single chain antibodies, e.g.,single chain Fv in which the heavy and light chain variable domains arejoined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody.” The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Standard techniques of molecular biology may be used to prepare DNAsequences encoding the antibodies or antibody fragments of the presentinvention. Desired DNA sequences may be synthesized completely or inpart using oligonucleotide synthesis techniques. Site-directedmutagenesis and polymerase chain reaction (PCR) techniques may be usedas appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)2 fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g., mammalian, host cell expression systems may be usedfor production of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include, but are not limitedto, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector encoding a nucleic acid of thepresent invention under conditions suitable for expression of proteinfrom DNA encoding the antibody molecule of the present invention, andisolating the antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced by(i) expressing a nucleic acid sequence according to the invention in ahost cell, and (ii) isolating the expressed antibody product.Additionally, the method may include (iii) purifying the isolatedantibody.

Transformed B cells and cultured plasma cells may be screened for thoseproducing antibodies of the desired specificity or function.

The screening step may be carried out by any immunoassay, e.g., ELISA,by staining of tissues or cells (including transfected cells), byneutralization assay or by one of a number of other methods known in theart for identifying desired specificity or function. The assay mayselect on the basis of simple recognition of one or more antigens, ormay select on the additional basis of a desired function e.g., to selectneutralizing antibodies rather than just antigen-binding antibodies, toselect antibodies that can change characteristics of targeted cells,such as their signaling cascades, their shape, their growth rate, theircapability of influencing other cells, their response to the influenceby other cells or by other reagents or by a change in conditions, theirdifferentiation status, etc.

Individual transformed B cell clones may then be produced from thepositive transformed B cell culture. The cloning step for separatingindividual clones from the mixture of positive cells may be carried outusing limiting dilution, micromanipulation, single cell deposition bycell sorting or another method known in the art.

Nucleic acid from the cultured plasma cells can be isolated, cloned andexpressed in HEK293T cells or other known host cells using methods knownin the art.

The immortalized B cell clones or the transfected host-cells of theinvention can be used in various ways e.g., as a source of monoclonalantibodies, as a source of nucleic acid (DNA or mRNA) encoding amonoclonal antibody of interest, for research, etc.

The invention provides a composition comprising immortalized B memorycells or transfected host cells that produce antibodies that neutralizeinfection of both that neutralizes infection of RSV, MPV and PVM.

The immortalized B cell clone or the cultured plasma cells of theinvention may also be used as a source of nucleic acid for the cloningof antibody genes for subsequent recombinant expression. Expression fromrecombinant sources is more common for pharmaceutical purposes thanexpression from B cells or hybridomas e.g., for reasons of stability,reproducibility, culture ease, etc.

Thus the invention provides a method for preparing a recombinant cell,comprising the steps of: (i) obtaining one or more nucleic acids (e.g.,heavy and/or light chain mRNAs) from the B cell clone or the culturedplasma cells that encodes the antibody of interest; (ii) inserting thenucleic acid into an expression vector and (iii) transfecting the vectorinto a host cell in order to permit expression of the antibody ofinterest in that host cell.

Similarly, the invention provides a method for preparing a recombinantcell, comprising the steps of: (i) sequencing nucleic acid(s) from the Bcell clone or the cultured plasma cells that encodes the antibody ofinterest; and (ii) using the sequence information from step (i) toprepare nucleic acid(s) for insertion into a host cell in order topermit expression of the antibody of interest in that host cell. Thenucleic acid may, but need not, be manipulated between steps (i) and(ii) to introduce restriction sites, to change codon usage, and/or tooptimize transcription and/or translation regulatory sequences.

The invention also provides a method of preparing a transfected hostcell, comprising the step of transfecting a host cell with one or morenucleic acids that encode an antibody of interest, wherein the nucleicacids are nucleic acids that were derived from an immortalized B cellclone or a cultured plasma cell of the invention. Thus the proceduresfor first preparing the nucleic acid(s) and then using it to transfect ahost cell can be performed at different times by different people indifferent places (e.g., in different countries).

These recombinant cells of the invention can then be used for expressionand culture purposes. They are particularly useful for expression ofantibodies for large-scale pharmaceutical production. They can also beused as the active ingredient of a pharmaceutical composition. Anysuitable culture technique can be used, including but not limited tostatic culture, roller bottle culture, ascites fluid, hollow-fiber typebioreactor cartridge, modular minifermenter, stirred tank, microcarrierculture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cellsor plasma cells are well known in the art (e.g., see Chapter 4 of KubyImmunology, 4th edition, 2000).

The transfected host cell may be a eukaryotic cell, including yeast andanimal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells,human cells such as PER.C6 or HKB-11 cells, myeloma cells), as well asplant cells. Preferred expression hosts can glycosylate the antibody ofthe invention, particularly with carbohydrate structures that are notthemselves immunogenic in humans. In one embodiment the transfected hostcell may be able to grow in serum-free media. In a further embodimentthe transfected host cell may be able to grow in culture without thepresence of animal-derived products. The transfected host cell may alsobe cultured to give a cell line.

The invention also provides a method for preparing one or more nucleicacid molecules (e.g., heavy and light chain genes) that encode anantibody of interest, comprising the steps of: (i) preparing animmortalized B cell clone or culturing plasma cells according to theinvention; (ii) obtaining from the B cell clone or the cultured plasmacells nucleic acid that encodes the antibody of interest. Further, theinvention provides a method for obtaining a nucleic acid sequence thatencodes an antibody of interest, comprising the steps of: (i) preparingan immortalized B cell clone or culturing plasma cells according to theinvention; (ii) sequencing nucleic acid from the B cell clone or thecultured plasma cells that encodes the antibody of interest.

The invention also provides a method of preparing nucleic acidmolecule(s) that encode an antibody of interest, comprising the step ofobtaining the nucleic acid that was obtained from a transformed B cellclone or cultured plasma cells of the invention. Thus the procedures forfirst obtaining the B cell clone or the cultured plasma cell, and thenobtaining nucleic acid(s) from the B cell clone or the cultured plasmacells can be performed at different times by different people indifferent places (e.g., in different countries).

The invention provides a method for preparing an antibody (e.g., forpharmaceutical use), comprising the steps of: (i) obtaining and/orsequencing one or more nucleic acids (e.g., heavy and light chain genes)from the selected B cell clone or the cultured plasma cells expressingthe antibody of interest; (ii) inserting the nucleic acid(s) into orusing the nucleic acid(s) sequence(s) to prepare an expression vector;(iii) transfecting a host cell that can express the antibody ofinterest; (iv) culturing or sub-culturing the transfected host cellsunder conditions where the antibody of interest is expressed; and,optionally, (v) purifying the antibody of interest.

The invention also provides a method of preparing an antibody comprisingthe steps of: culturing or sub-culturing a transfected host cellpopulation under conditions where the antibody of interest is expressedand, optionally, purifying the antibody of interest, wherein saidtransfected host cell population has been prepared by (i) providingnucleic acid(s) encoding a selected antibody of interest that isproduced by a B cell clone or cultured plasma cells prepared asdescribed above, (ii) inserting the nucleic acid(s) into an expressionvector, (iii) transfecting the vector in a host cell that can expressthe antibody of interest, and (iv) culturing or sub-culturing thetransfected host cell comprising the inserted nucleic acids to producethe antibody of interest. Thus the procedures for first preparing therecombinant host cell and then culturing it to express antibody can beperformed at very different times by different people in differentplaces (e.g., in different countries).

Epitopes

As mentioned above, the antibodies of the invention can be used to mapthe epitopes to which they bind. The inventors have discovered that theneutralizing antibodies of the invention are directed towards epitopesfound on the pre-fusion, but not post-fusion, F protein. In oneembodiment, the antibodies, or antigen binding fragments thereof, bindRSV pre-fusion F protein and not RSV post-fusion F protein. In anotherembodiment, the antibodies, or antigen binding fragments thereof, bindthe pre-fusion F protein and not the post-fusion F protein of RSV andMPV. In yet another embodiment, the antibodies, or antigen bindingfragments thereof, bind to the pre-fusion F protein but not to thepost-fusion F protein of RSV, MPV and PVM.

The epitopes to which the antibodies of the invention bind may be linear(continuous) or conformational (discontinuous). In one embodiment, theantibodies and antibody fragments of the invention bind a conformationalepitope. In another embodiment, the conformational epitope is presentonly under non-reducing conditions. Without being bound to any theory,the conformational epitope bound by the antibodies of the inventionrelies on the presence of disulphide bonds between amino acid residueson the F protein.

In another embodiment, the epitope to which the antibodies of theinvention bind is distinct from antigenic site I, antigenic site II,antigenic site IV as defined on the RSV post-fusion F protein andcorresponding sites on the MPV F protein. In yet another embodiment, theantibodies and antigen binding fragments of the invention do notcross-compete with Palivizumab, Motavizumab, mAb 101F, mAb 131-2A or mAbD25 for binding to the F protein of RSV; nor do they cross-compete withmAb 234 for binding to the F protein of MPV.

In another embodiment, the region to which the antibodies of theinvention bind comprises a polypeptide located in the N-terminal portionof the RSV F protein, spanning residues SAVSKGYLSALRTGWYTSVIT (SEQ IDNO: 63). The core part in this polypeptide is formed by the residuesY(x1)S(x2)LRTGW (SEQ ID NO:70), which are highly conserved between RSV,MPV and PVM, and wherein the amino acid at position (x1) can be, but isnot limited to, L, F, or K, and wherein amino acid at position (x2) canbe, but is not limited to, A or V. Examples of polypeptide variants towhich the antibodies of the invention bind include, but are not limitedto, YLSALRTGW (SEQ ID NO: 64), YLSVLRTGW (SEQ ID NO: 65), YFSALRTGW (SEQID NO: 66), YFSVLRTGW (SEQ ID NO: 67), YKSALRTGW (SEQ ID NO: 68), andYKSVLRTGW (SEQ ID NO: 69).

The polypeptides that bind to the antibodies of the present inventionmay have a number of uses. The polypeptides and polypeptide variantsthereof in purified or synthetic form can be used to raise immuneresponses (i.e., as a vaccine, or for the production of antibodies forother uses) or for screening sera for antibodies that immunoreact withthe epitope or mimotopes thereof. In one embodiment such polypeptides orpolypeptide variants, or antigen comprising such an polypeptides orpolypeptide variants may be used as a vaccine for raising an immuneresponse that comprises antibodies of the same quality as thosedescribed in the present invention. The antibodies and antibodyfragments of the invention can also be used in a method of monitoringthe quality of vaccines. In particular the antibodies can be used tocheck that the antigen in a vaccine contains the correct immunogenicepitope in the correct conformation. The use of an antibody of theinvention, or an antigen binding fragment thereof, for monitoring thequality of a vaccine against RSV or MPV or both RSV and MPV by, forexample, checking that the antigen of said vaccine contains the specificepitope in the correct conformation is also contemplated to be withinthe scope of the invention.

The polypeptides that bind to the antibodies of the present inventionmay also be useful in screening for ligands that bind to saidpolypeptides. Such ligands, include but are not limited to antibodies;including those from camels, sharks and other species, fragments ofantibodies, peptides, phage display technology products, aptamers,adnectins or fragments of other viral or cellular proteins, may blockthe epitope and so prevent infection. Such ligands are encompassedwithin the scope of the invention.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition comprising one ormore of: the antibodies or antibody fragments of the invention; nucleicacid encoding such antibodies or fragments; vectors encoding the nucleicacids; or polypeptides recognized by the antibodies or antigen bindingfragment of the invention. The pharmaceutical composition may alsocontain a pharmaceutically acceptable carrier or excipient. Although thecarrier or excipient may facilitate administration, it should not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Nor should it be toxic. Suitable carriers may be large,slowly metabolized macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the subject.

Within the scope of the invention are compositions present in severalforms of administration; the forms include, but are not limited to,those forms suitable for parenteral administration, e.g., by injectionor infusion, for example by bolus injection or continuous infusion.Where the product is for injection or infusion, it may take the form ofa suspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilizing and/or dispersing agents. Alternatively, the antibodymolecule may be in dry form, for reconstitution before use with anappropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. In one embodiment the compositions are adaptedfor administration to mammalian, e.g., human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Typically, the therapeutic compositionsmay be prepared as injectables, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule. Knownantibody-based pharmaceuticals provide guidance relating to frequency ofadministration e.g., whether a pharmaceutical should be delivered daily,weekly, monthly, etc. Frequency and dosage may also depend on theseverity of symptoms.

Compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g., a lyophilized composition, like Synagis™ and Herceptin™,for reconstitution with sterile water containing a preservative). Thecomposition may be prepared for topical administration e.g., as anointment, cream or powder. The composition may be prepared for oraladministration e.g., as a tablet or capsule, as a spray, or as a syrup(optionally flavored). The composition may be prepared for pulmonaryadministration e.g., as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g., asdrops. The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a subject.For example, a lyophilized antibody can be provided in kit form withsterile water or a sterile buffer.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule, an antibody fragment or variants andderivatives thereof. As such, it will be susceptible to degradation inthe gastrointestinal tract. Thus, if the composition is to beadministered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between5.5 and 8.5, in some embodiments this may be between 6 and 8, and inother embodiments about 7. The pH may be maintained by the use of abuffer. The composition may be sterile and/or pyrogen free. Thecomposition may be isotonic with respect to humans. In one embodimentpharmaceutical compositions of the invention are supplied inhermetically-sealed containers.

Pharmaceutical compositions will include an effective amount of one ormore antibodies of the invention and/or a polypeptide comprising anepitope that binds an antibody of the invention i.e., an amount that issufficient to treat, ameliorate, attenuate or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic effect. Therapeuticeffects also include reduction or attenuation in pathogenic potency orphysical symptoms. The precise effective amount for any particularsubject will depend upon their size, weight, and health, the nature andextent of the condition, and the therapeutics or combination oftherapeutics selected for administration. The effective amount for agiven situation is determined by routine experimentation and is withinthe judgment of a clinician. For purposes of the present invention, aneffective dose will generally be from about 0.01 mg/kg to about 50mg/kg, or about 0.05 mg/kg to about 10 mg/kg of the compositions of thepresent invention in the individual to which it is administered. Knownantibody-based pharmaceuticals provide guidance in this respect e.g.,Herceptin™ is administered by intravenous infusion of a 21 mg/mlsolution, with an initial loading dose of 4 mg/kg body weight and aweekly maintenance dose of 2 mg/kg body weight; Rituxan™ is administeredweekly at 375 mg/m²; etc.

In one embodiment compositions can include more than one (e.g., 2, 3,etc.) antibodies of the invention to provide an additive or synergistictherapeutic effect. In another embodiment, the composition may compriseone or more (e.g., 2, 3, etc.) antibodies of the invention and one ormore (e.g., 2, 3, etc.) additional antibodies against RSV, MPV or bothRSV and MPV. Further, the administration of antibodies of the inventiontogether with antibodies specific to other pathogens, for example,influenza A or influenza B virus, are within the scope of the invention.The antibodies of the invention can be administered eithercombined/simultaneously or at separate times from antibodies of specificto pathogens other than RSV or MPV.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is an antibody of the invention as described herein and thesecond antibody is specific for RSV, MPV or both RSV and MPV or adifferent pathogen that may have co-infected the subject to whom thepharmaceutical composition is being administered.

Examples of antibodies of the invention specific for, and thatneutralize RSV, MPV and PVM include, but are not limited to, HMB3210variant 3, HMB3210 variant 1, HMB3210 variant 2, HMB3210 variant 4,HMB3210 variant 5, HMB3210 variant 6, HMB2430 variant 1, HMB2430 variant2, HMB2430 variant 3, HMB2430 variant 4 or HMB2430 variant 5.

In one embodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB3210 variant 1 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB3210 variant 2 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB3210 variant 3 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB3210 variant 4 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB3210 variant 5 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB3210 variant 6 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising the antibody HMB2430 variant 1 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier. Inanother embodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB2430 variant 2 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB2430 variant 3 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB2430 variant 4 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody HMB2430 variant 5 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier.

Antibodies of the invention may be administered (either combined orseparately) with other therapeutics e.g., with chemotherapeuticcompounds, with radiotherapy, etc. In one embodiment, the therapeuticcompounds include anti-viral compounds such as Tamiflu™. Suchcombination therapy provides an additive or synergistic improvement intherapeutic efficacy relative to the individual therapeutic agents whenadministered alone. The term “synergy” is used to describe a combinedeffect of two or more active agents that is greater than the sum of theindividual effects of each respective active agent. Thus, where thecombined effect of two or more agents results in “synergisticinhibition” of an activity or process, it is intended that theinhibition of the activity or process is greater than the sum of theinhibitory effects of each respective active agent. The term“synergistic therapeutic effect” refers to a therapeutic effect observedwith a combination of two or more therapies wherein the therapeuticeffect (as measured by any of a number of parameters) is greater thanthe sum of the individual therapeutic effects observed with therespective individual therapies.

Antibodies may be administered to those subjects who have previouslyshown no response, i.e., have been shown to be refractive to treatmentfor RSV or MPV infection. Such treatment may include previous treatmentwith an anti-viral agent. This may be due to, for example, infectionwith an anti-viral resistant strain of RSV, MPV or both RSV and MPV.

In one embodiment, a composition of the invention may include antibodiesof the invention, wherein the antibodies may make up at least 50% byweight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more)of the total protein in the composition. In such a composition, theantibodies are in purified form.

The invention provides a method of preparing a pharmaceuticalcomposition comprising the steps of: (i) preparing an antibody of theinvention; and (ii) admixing the purified antibody with one or morepharmaceutically-acceptable carriers.

In another embodiment, a method of preparing a pharmaceuticalcomposition comprises the step of: admixing an antibody with one or morepharmaceutically-acceptable carriers, wherein the antibody is amonoclonal antibody that was obtained from a transformed B cell or acultured plasma cell of the invention. Thus the procedures for firstobtaining the monoclonal antibody and then preparing the pharmaceuticalcan be performed at very different times by different people indifferent places (e.g., in different countries).

As an alternative to delivering antibodies or B cells for therapeuticpurposes, it is possible to deliver nucleic acid (typically DNA) thatencodes the monoclonal antibody (or active fragment thereof) of interestderived from the B cell or the cultured plasma cells to a subject, suchthat the nucleic acid can be expressed in the subject in situ to providea desired therapeutic effect. Suitable gene therapy and nucleic aciddelivery vectors are known in the art.

Compositions of the invention may be immunogenic compositions, and insome embodiments may be vaccine compositions comprising an antigencomprising an epitope recognized by an antibody of the invention or anantigen binding fragment thereof. Vaccines according to the inventionmay either be prophylactic (i.e., prevent infection) or therapeutic(i.e., treat or ameliorate infection).

Compositions may include an antimicrobial, particularly if packaged in amultiple dose format. They may comprise detergent e.g., a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g., less than 0.01%. Compositions may also include sodium salts(e.g., sodium chloride) to give tonicity. A concentration of 10±2 mg/mlNaCl is typical.

Further, compositions may comprise a sugar alcohol (e.g., mannitol) or adisaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg/ml(e.g., 25 mg/ml), particularly if they are to be lyophilized or if theyinclude material which has been reconstituted from lyophilized material.The pH of a composition for lyophilisation may be adjusted to between 5and 8, or between 5.5 and 7, or around 6.1 prior to lyophilisation.

The compositions of the invention may also comprise one or moreimmunoregulatory agents. In one embodiment, one or more of theimmunoregulatory agents include(s) an adjuvant.

The epitope compositions of the invention may elicit both a cellmediated immune response as well as a humoral immune response in orderto effectively address RSV and MPV infection. This immune response mayinduce long lasting (e.g., neutralizing) antibodies and a cell mediatedimmunity that can quickly respond upon exposure to RSV or MPV or bothRSV and MPV.

Medical Treatments and Uses

The antibodies and antibody fragments of the invention or derivativesand variants thereof may be used for the treatment of RSV or MPVinfection or co-infection with both RSV and MPV; for the prevention ofinfection of RSV or MPV or both RSV and MPV; or for the diagnosis of RSVor MPV infection.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be tissue samples taken from,for example, nasal passages, sinus cavities, salivary glands, lung,liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart,ovaries, pituitary, adrenals, thyroid, brain or skin. The methods ofdiagnosis may also include the detection of an antigen/antibody complex.

The invention therefore provides (i) an antibody, an antibody fragment,or variants and derivatives thereof according to the invention, (ii) animmortalized B cell clone according to the invention, (iii) an epitopecapable of binding an antibody of the invention or (iv) a ligand,preferably an antibody, capable of binding an epitope that binds anantibody of the invention for use in therapy.

The invention also provides a method of treating a subject comprisingadministering to the subject an antibody, an antibody fragment, orvariants and derivatives thereof according to the invention, or, aligand, preferably an antibody, capable of binding an epitope that bindsan antibody of the invention. In one embodiment, the method results inreduced RSV or MPV infection in the subject. In another embodiment, themethod prevents, reduces the risk or delays of RSV or MPV infection inthe subject.

The invention also provides the use of (i) an antibody, an antibodyfragment, or variants and derivatives thereof according to theinvention, (ii) an immortalized B cell clone according to the invention,(iii) an epitope capable of binding an antibody of the invention, (iv) aligand, preferably an antibody, that binds to an epitope capable ofbinding an antibody of the invention, or (v) a pharmaceuticalcomposition of the invention in (i) the manufacture of a medicament forthe treatment or attenuation of infection by RSV or MPV or both RSV andMPV, (ii) a vaccine, or (iii) diagnosis of RSV and MPV infection.

The invention provides a composition of the invention for use as amedicament for the prevention or treatment of RSV or MPV infection. Italso provides the use of an antibody of the invention and/or a proteincomprising an epitope to which such an antibody binds in the manufactureof a medicament for treatment of a subject and/or diagnosis in asubject. It also provides a method for treating a subject, comprisingthe step of administering to the subject a composition of the invention.In some embodiments the subject may be a human. One way of checkingefficacy of therapeutic treatment involves monitoring disease symptomsafter administration of the composition of the invention. Treatment canbe a single dose schedule or a multiple dose schedule.

In one embodiment, an antibody, antibody fragment, immortalized B cellclone, epitope or composition according to the invention is administeredto a subject in need of such treatment. Such a subject includes, but isnot limited to, one who is particularly at risk of or susceptible to RSVor MPV infection, including, for example, an immunocompromised subject.The antibody or antibody fragment of the invention can also be used inpassive immunization or active vaccination.

Antibodies and fragments thereof as described in the present inventionmay also be used in a kit for the diagnosis of RSV or MPV infection.Further, epitopes capable of binding an antibody of the invention may beused in a kit for monitoring the efficacy of vaccination procedures bydetecting the presence of protective anti-RSV or anti-MPV antibodies.Antibodies, antibody fragment, or variants and derivatives thereof, asdescribed in the present invention may also be used in a kit formonitoring vaccine manufacture with the desired immunogenicity.

The invention also provides an epitope that specifically binds to anantibody of the invention or an antigen binding fragment thereof, foruse (i) in therapy, (ii) in the manufacture of a medicament for thetreatment or attenuation of RSV or MPV or both RSV and MPV infection,(iii) as a vaccine, or (iv) in screening for ligands able to neutralizeRSV or MPV or both RSV and MPV infection.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing a monoclonal antibody with one or morepharmaceutically-acceptable carriers, wherein the monoclonal antibody isa monoclonal antibody that was obtained from a transfected host cell ofthe invention. Thus the procedures for first obtaining the monoclonalantibody (e.g., expressing it and/or purifying it) and then admixing itwith the pharmaceutical carrier(s) can be performed at very differenttimes by different people in different places (e.g., in differentcountries).

Starting with a transformed B cell or a cultured plasma cell of theinvention, various steps of culturing, sub-culturing, cloning,sub-cloning, sequencing, nucleic acid preparation etc. can be performedin order to perpetuate the antibody expressed by the transformed B cellor the cultured plasma cell, with optional optimization at each step. Inone embodiment, the above methods further comprise techniques ofoptimization (e.g., affinity maturation or optimization) applied to thenucleic acids encoding the antibody. The invention encompasses allcells, nucleic acids, vectors, sequences, antibodies etc. used andprepared during such steps.

In all these methods, the nucleic acid used in the expression host maybe manipulated to insert, delete or alter certain nucleic acidsequences. Changes from such manipulation include, but are not limitedto, changes to introduce restriction sites, to amend codon usage, to addor optimize transcription and/or translation regulatory sequences, etc.It is also possible to change the nucleic acid to alter the encodedamino acids. For example, it may be useful to introduce one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions,deletions and/or insertions into the antibody's amino acid sequence.Such point mutations can modify effector functions, antigen-bindingaffinity, post-translational modifications, immunogenicity, etc., canintroduce amino acids for the attachment of covalent groups (e.g.,labels) or can introduce tags (e.g., for purification purposes).Mutations can be introduced in specific sites or can be introduced atrandom, followed by selection (e.g., molecular evolution). For instance,one or more nucleic acids encoding any of the CDR regions, heavy chainvariable regions or light chain variable regions of antibodies of theinvention can be randomly or directionally mutated to introducedifferent properties in the encoded amino acids. Such changes can be theresult of an iterative process wherein initial changes are retained andnew changes at other nucleotide positions are introduced. Further,changes achieved in independent steps may be combined. Differentproperties introduced into the encoded amino acids may include, but arenot limited to, enhanced affinity.

General

As used herein, the terms “antigen binding fragment,” “fragment,” and“antibody fragment” are used interchangeably to refer to any fragment ofan antibody of the invention that retains the antigen-binding activityof the antibody. Examples of antibody fragments include, but are notlimited to, a single chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv.Further, the term “antibody” as used herein includes both antibodies andantigen binding fragments thereof.

As used herein, a “neutralizing antibody” is one that can neutralize,i.e., prevent, inhibit, reduce, impede or interfere with, the ability ofa pathogen to initiate and/or perpetuate an infection in a host. Theterms “neutralizing antibody” and “an antibody that neutralizes” or“antibodies that neutralize” are used interchangeably herein. Theseantibodies can be used alone, or in combination, as prophylactic ortherapeutic agents upon appropriate formulation, in association withactive vaccination, as a diagnostic tool, or as a production tool asdescribed herein.

The term “comprising” encompasses “including” as well as “consisting”e.g., a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g., X+Y.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means, for example,x±5%, or x±7%, or x±10%, or x±12%, or x±15%, or x±20%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

As used herein, reference to “treatment” of a subject or patient isintended to include prevention, prophylaxis, attenuation, ameliorationand therapy. The terms “subject” or “patient” are used interchangeablyherein to mean all mammals including humans. Examples of subjectsinclude humans, cows, dogs, cats, horses, goats, sheep, pigs, andrabbits. In one embodiment, the patient is a human.

EXAMPLES

Exemplary embodiments of the present invention are provided in thefollowing examples. The following examples are presented only by way ofillustration and to assist one of ordinary skill in using the invention.The examples are not intended in any way to otherwise limit the scope ofthe invention.

Example 1. Isolation and Characterization of Monoclonal Antibodies fromHuman Memory B Cells Able to Cross-Neutralize Both RSV and MPV

From a cohort of 125 blood donors we selected 7 donors showing highserum antibody titers against RSV and MPV. CD22+ IgG+ B cells weresorted from cryopreserved peripheral blood mononuclear cells (PBMCs) andimmortalized at 3 to 5 cells/well using Epstein Barr Virus (EBV) and CpGoligodeoxynucleotide 2006 and irradiated allogeneic PBMCs as feedercells. Culture supernatants were harvested after 14 days and analyzedfor the presence of neutralizing antibodies using a microneutralizationassay based on infection of Hep-2 cells by RSV strain A2 or of LLC-MK2cells by MPV A1 I-PV-03/01-6621 strain. Neat supernatants were incubatedwith 50-100 TCID₅₀ of viruses for 1 hour at room temperature prior toaddition of Hep-2 or LLC-MK2 target cells which were incubated for 6 or8 days, respectively. Viable cells were then detected with aspectrophotometer by adding to the cultures the WST-1 reagent (Roche)for 3 to 4 hours.

From three independent experiments, 36 monoclonal antibodies (mAbs) thatneutralized MPV (FIG. 1, left panel) were isolated; and from fiveindependent experiments, 136 mAbs that neutralized RSV (FIG. 1, rightpanel) were isolated. A secondary screening was then performed to testwhether the isolated mAbs were able to neutralize both WV and RSV. Usingthis strategy two mAbs isolated from the same donor (Don. 5) were foundto cross-neutralize RSV and MPV: (i) HMB2430, which was initiallyselected based on neutralization of RSV, and (ii) HMB3210, which wasinitially selected based on neutralization of MPV.

The VH and VL genes of HMB2430 and HMB3210 were cloned into IgG1expression vectors and recombinant mAbs were produced by transienttransfection of 293 Freestyle cells (293F). Supernatants fromtransfected cells were collected after 10 days of culture and IgG wereaffinity purified by Protein A chromatography. The two mAbs shared mostV and J gene fragments (IGHV3-21*01, IGHJ4*02, IGLV1-40*01 andIGLJ1*01), according to the homology analysis performed using the IMGTdatabase, but differed in the N regions in the IGHD usage (D3-10*01 andD5-24*01 for HMB2430 and HMB3210, respectively) and in the pattern ofsomatic mutations, and were therefore considered not clonally related.

The half maximal inhibitory concentration (IC50) of HMB2430 and HMB3210was determined using the microneutralization assay described above with100 tissue culture infective dose 50 (TCID50) of virus. IC50 values werecalculated by interpolation of neutralization curves fitted with a4-parameter nonlinear regression with a variable slope. The results ofthe analysis are shown in FIG. 2 and Table 4.

TABLE 4 mAbs IC50 (ng/ml) Viruses (group) HMB2430 HMB3210 Palivizumab234 RSV A2 (A) 146 86 524 >20000 MPV I-PV 03/01-6621 (A1) 393 52 >200007

Example 2. Breadth of Reactivity to all RSV and MPV Groups andSub-Groups

In order to evaluate the breath of reactivity of HMB2430 and HMB3210,purified mAbs were tested by FACS for binding to Hep-2 RSV-infectedcells or to LLC-MK2 MPV-infected cells using the following RSV and MPVstrains: RSV A2 (A, 1961 Australia; A/A2/61), RSV Long (A, Maryland US,1956; A/Long/56), RSV Randall (A, Chicago US, 1958; A/Randall/58), RSV9320 (B, Massachusetts US, 1977; B/9320/77), WV/14617/85 (B, HuntingtonW. Va., 1985; B/14617/85), 18537 (B, Washington D.C. US, 1962;B/18537/62), WV I-PV-03/01-6621 (A1, Pavia IT, 2001; A1/6621/01), MPVI-PV-02/06-8938 (A2, Pavia IT, 2006; A2/8938/06), I-PV-03/04-4702 (B1,Pavia IT, 2004; B1/4702/04) and I-PV-02/04-3817 (B2, Pavia IT, 2004;B2/3817/04). In parallel, three previously described mAbs were tested:(i) Motavizumab, RSV-specific; (ii) mAb 234, MPV-specific; and (iii)FO32, Influenza A-specific (used as negative control). All mAbs weretested for binding to infected or uninfected cells at 10 μg/ml. HMB2430and HMB3210 reacted with all 6 RSV and all 4 WV strains tested,representative of the known RSV and WV groups and sub-groups (Table 5).In contrast, Motavizumab reacted with all the 6 RSV strains tested, butdid not react with any of the 4 WV strains tested. Conversely, 234 mAbreacted will all 4 WV strains tested but did not react with any of the 6RSV strains tested.

TABLE 5 Staining of Hep-2 or LLC-MK2 cells infected by different strainsof RSV or MIN, respectively, by FO32 (negative control), Motavizumab,234, HMB2430 and HMB3210, as measured by flow cytometry. MonoclonalAntibody (10 μg/ml) Virus FO32 Motavizumab mAb 234 HMB2430 HMB3210 RSVA/A2/61 − + − + + RSV A/Long/56 − + − + + RSV A/Randall/58 − + − + + RSVB/18537/62 − + − + + RSV B/14617/85 − + − + + RSV B/9320/77 − + − + +MPV A1/6621/01 − − + + + MPV A2/8938/06 − − + + + MPV B1/4702/04 −− + + + MPV B2/3817/04 − − + + + Mock LLC-MK2 − − − − − Mock Hep-2 − − −− − (−) <5% stained cells (+) >50% stained cells

Example 3. Binding to Recombinant F Protein of RSV and MPV by ELISA andby Staining of Transfected Cells

In order to identify the target antigen recognized by HMB2430 andHMB3210 on RSV and MPV viruses, we analyzed the two mAbs in parallelwith Motavizumab and mAb 234, for their ability to bind to ahomotrimeric soluble F protein of RSV (A2 strain) that was produced fromtransiently transfected 293F cells. As shown in FIG. 3, both HMB2430 andHMB3210 reacted specifically with RSV F protein by ELISA and showed adistinct binding profile as compared to Motavizumab. In addition, HM2430and HMB3210 stained intracellularly 293F cells transiently transfectedwith mammalian expression vectors encoding for the full length F proteinfrom either RSV (A2 strain) or MPV (NL/1/99 B1 strain) (Table 6),indicating that HMB2430 and HMB3210 recognize a shared epitope presenton both RSV and MPV F proteins. This finding is particularly strikingconsidering that RSV and MPV F proteins have only 33-35% amino acidsequence identity. As expected Palivizumab and Motavizumab bound tocells expressing the RSV F protein but not to those expressing the MPV Fprotein (Table 6). Conversely, 234 mAb bound to cells expressing the MPVF protein but not to cells expressing the RSV F protein (Table 6).

TABLE 6 Staining of untransfected 293F cells or 293F cells transfectedwith RSV or MPV F protein, as measured by flow cytometry Antibody 293F +293F + 293F (10 μg/ml) RSV F (A/A2/61;A) MPV F (NL/1/99;B1)untransfected HMB3210 + + − HMB2410 + + − Palivizumab + − −Motavizumab + − − 234 − + − (−) <5% stained cells (+) >50% stained cells

Example 4. Epitope Mapping by Using an Inhibition Binding Assay on RSVInfected Cells

In order to gain insight into the F protein epitope recognized byHMB2430 and HMB3210, we set up an inhibition of binding assay usingHep-2 cells infected with RSV A2 strain. The following panel of RSV Fprotein-specific mAbs were purchased or produced by gene synthesis: (i)Motavizumab, specific for the antigenic site II; (ii) 101F, specific forthe antigenic site IV; (iii) D25 of undefined specificity; (iv) 131-2a,specific for the antigenic site I. The mAbs were labeled with biotin andtested for binding to Hep-2 infected cells to determine the optimalconcentration of mAb required to achieve 70-80% maximal binding. Thebiotin-labeled mAbs were then used as probes to assess whether theirbinding (measured using fluorophore-conjugated streptavidin) wasinhibited by pre-incubation of RSV A2-infected cells with a 50 foldexcess of homologous or heterologous unlabeled mAbs. As expected,binding of biotin-labeled HMB2430 was blocked by preincubating the cellswith unlabeled HMB2430, but it was also partially blocked by unlabeledHMB3210 (FIG. 4). In contrast, binding of all the other biotin-labeledmAbs tested was not prevented by pre-incubation with either HMB2430 orHMB3210 (FIG. 4). Taken together these results indicate that HMB2430 andHMB3210 recognize partially overlapping epitopes on the F protein thatare shared in RSV and WV and that these epitopes are distinct from theepitopes in the F protein antigenic site II (recognized by Motavizumaband Palivizumab), antigenic site IV (recognized by mAb 101F), andantigenic site I (recognized by mAb 131-2A). In addition, the epitopesrecognized by mAbs HMB2430 and HMB3210 on the RSV F protein are distinctfrom the unknown epitope recognized by the mAb D25.

Example 5. Monoclonal Antibody Reactivity with the RSV and MPV FProteins Under Reducing and Non-Reducing Conditions

To further confirm the finding that HMB2430 and HMB3210 recognize the Fprotein of both RSV and MPV, we tested the two mAbs for their ability tostain RSV and WV F proteins in Western blot. Hep-2 cells were infectedwith RSV and LLC-MK2 cells with MPV, lysed with a mild detergent and runon SDS-PAGE gel under reducing or non-reducing conditions. Proteins werethen transferred on a PVDF membrane which was then incubated with eitherHMB2430, HMB3210, Motavizumab or 234 mAb. MAb binding was detected withan anti-human HRP-conjugated antibody in combination with the ECLWestern Blotting Detection reagent. HMB2430 and HMB3210 bound to Fprotein derived from RSV-infected cells (FIG. 5) and MPV-infected cells(FIG. 6) under non-reducing conditions. The MPV-specific mAb 234 (thatrecognizes an epitope on the WV F protein which correspond to theantigenic site II on RSV F protein) bound to WV F protein undernon-reducing conditions, but did not bind to RSV F protein. In contrast,the RSV-specific mAb Motavizumab bound to the RSV F protein both underreducing and non-reducing conditions, confirming the recognition of alargely linear epitope. These results suggest that, differently fromMotavizumab and Palivizumab, HMB2430 and HMB3210 recognizeconformational epitopes which also relies on the presence of disulphidebonds between amino acid residues on the RSV and WV F proteins.

Example 6. Neutralization of all RSV and MPV Groups and Sub-Groups byHMB2430 and HMB3210

Purified HMB2430 and HMB3210 mAbs were tested for their ability toneutralize RSV or MPV infection of Hep-2 or LLC-MK2 cells, respectively.The following RSV and MPV strains were tested: RSV A2 (A, 1961Australia; A/A2/61), RSV Long (A, Maryland US, 1956; A/Long/56), RSVRandall (A, Chicago US, 1958; A/Randall/58), RSV 9320 (B, MassachusettsUS, 1977; B/9320/77), WV/14617/85 (B, Huntington W. Va., 1985;B/14617/85), 18537 (B, Washington D.C. US, 1962; B/18537/62), RSV9727/2009 (B, Pavia IT, 2009; B/9727/09), RSV 9736/2009 (B, Pavia IT,2009; B/9736/09), RSV 9847/2009 (B, Pavia IT, 2009; B/9847/09), MPVI-PV-03/01-6621 (A1, Pavia IT, 2001; A1/6621/01), MPV I-PV-02/06-8938(A2, Pavia IT, 2006; A2/8938/06), I-PV-02/06-8908 (A2, Pavia IT, 2006;A2/8908/06), I-PV-02/06-8909 (A2, Pavia IT, 2006; A2/8909/06),I-PV-03/04-4702 (B1, Pavia IT, 2004; B1/4702/04) and I-PV-02/04-3817(B2, Pavia IT, 2004; B2/3817/04).

In the same experiment HMB2430 and HMB3210 were compared to Palivizumab(RSV-specific) and 234 mAb (MPV-specific). HMB3210 neutralized all 11RSV and all 6 MPV strains tested, representative of the known RSV and WVgroups and sub-groups (Table 7). HMB2430 neutralized all 11 RSV strainstested and all the A1 and A2 MPV strains tested but not the B1 or B2 MPVstrains tested. As expected, Palivizumab neutralized all the 11 RSVstrains tested, but none of the 6 MPV strains tested while 234 mAbneutralized all 6 MPV strains tested but none of the 11 RSV strainstested.

HMB3210 and HMB2430 potently neutralized all 11 RSV strains tested (meanIC50 values, 0.070 and 0.133 μg/ml, respectively. These values were onaverage 5.4 and 2.6 fold higher than the IC50 value of Palivizumab(0.284 μg/ml). HMB3210 potently neutralized all 6 WV strains tested(IC50 mean value 0.113 μg/ml) that is on average 1.7 fold lower than theIC50 value of 234 (0.046 μg/ml).

TABLE 7 Neutralization of RSV and MPV strains Neutralization IC50(μg/ml) Virus Palivizumab mAb 234 HMB2430 HMB3210 RSV A/A2/61 0.617 —0.350 0.184 RSV A/Long/56 0.599 — 0.361 0.187 RSV A/Randall/58 0.440 —0.179 0.116 RSV A/9846/09 0.283 — 0.123 0.06 RSV A/9835/09 0.284 — 0.0760.063 RSV B/18537/62 0.143 — 0.094 0.034 RSV B/14617/85 0.129 — 0.0960.038 RSV B/9727/09 0.275 — 0.084 0.051 RSV B/9320/77 0.069 — 0.0210.012 RSV B/9736/09 0.092 — 0.027 0.007 RSV B/9847/09 0.209 — 0.0530.026 MPV A1/6621/01 — 0.040 0.744 0.071 MPV A2/8938/06 — 0.044 1.0490.045 MPV A2/8908/06 — 0.057 2.795 0.066 MPV A2/8909/06 — 0.012 0.1610.007 MPV B1/4702/04 — 0.019 — 0.029 MPV B2/3817/04 — 0.106 — 0.465

Example 7. Lack of Selection of RSV and MPV Viral Escape Mutants

HMB3210, Palivizumab and 234 mAb were tested for their ability to selectRSV or MPV Monoclonal Antibody Resistant Mutants (MARMs) in vitro. Inspite of several attempts, HMB3210 failed to select any RSV or MPV MARMswhen tested against 32×10e6 RSV A/Long/58 TCID50 and against 16×10e6 MPVA1/6621/01 TCID50. In contrast, Palivizumab selected MARMs with highfrequency (a total of 85 independent Palivizumab MARMs were isolatedfrom an input of 16×−10e6 RSV A/Long/58 TCID50 that corresponded to afrequency of 1 in 185,000 TCID50). MAb 234 under the same experimentalconditions did not select any MPV MARMs. The difficulty to isolate 234mAb MARMs is consistent with the report by Ulbrandt et al. (J GeneralVirol 2008) that showed that a high level of virus was required toisolate a small number of MARMs. Escaped viruses were mapped to amutation K242N using the NL/1/99 MPV B1 isolate. Independent PalivizumabMARMs (PZ-MARMs) were collected and the F protein of 10 of them wasfully sequenced (Table 8). PZ-MARM2, PZ-MARM3, PZ-MARM4, PZ-MARM5,PZ-MARM6, PZ-MARM8 and PZ-MARM10 shared the same two amino acidmutations (P101S/K272T); PZ-MARM1 had also two amino acid mutations inthe same position but with a different amino acid change (P101S/K272Q);PZ-MARM7 had a single amino acid mutation (K272N) again at position 272;finally, PZ-MARM9 had a mutation in common with other PZ-MARMs and aunique mutation at position 262 (P101S/N262Y). Point mutations in thisregion (nucleotide position 827 and 828) were already described (Zhao etal. Virology 2004) and resulted in two different amino acid changes atposition 272 (K272Q and K272M). The first mutation (i.e. K272Q) was alsopresent in the PZ-MARM1 here described. Viruses carrying these pointmutations were completely resistant to the prophylactic effects ofPalivizumab in cotton rats (Zhao et al. Virology 2004) and the samemutations along with others were described in RSV-infectedimmunosuppressed cotton rats treated prophylactically with Palivizumab.

HMB3210, HMB2430 and Palivizumab were then tested for their capacity toneutralize the PZ-MARM6 infection of Hep-2 cells. While Palivizumab didnot neutralize the PZ-MARM6, HMB3210 and HMB2430 potently neutralizedthis virus to levels comparable with those observed with thecorresponding wild type virus (FIG. 7).

Taken together, these results demonstrate that HMB3210 and HMB2430 didnot select RSV or MPV MARMs in vitro. These results are consistent withthe notion that the target epitopes recognized by HMB3210 and HMB2430are extremely conserved and that mutations that abrogate antibodybinding are either extremely rare or may be associated with loss ofviral fitness.

TABLE 8 Amino acid variations in RSVMARMs selected with Palivizumab aaposition (nucleotide position) 101 (314) 262 (797) 272 (827) 272 (828)272 (829) RSV A/Long/56 P N K K K PZ-MARM1 S (C to T) Q (A to C)PZ-MARM2 S (C to T) T (A to C) PZ-MARM3 S (C to T) T (A to C) PZ-MARM4 S(C to T) T (A to C) PZ-MARM5 S (C to T) T (A to C) PZ-MARM6 S (C to T) T(A to C) PZ-MARM7 P N (G to T) PZ-MARM8 S (C to T) T (A to C) PZ-MARM9 S(C to T) Y (A to T) PZ-MARM10 S (C to T) T (A to C)

Example 8. Removal of a Glycosylation Site in LCDR3 does not AffectHMB3210 Activity

HMB3210 variable light chain has a N-glycosylation motif (NxS/T, where xcan be any amino acid but proline) in the LCDR3 at position 113 (IMGTnumbering). The asparagine at position 113 (N113) replaces a serinepresent in the germline sequence. The presence of glycosylation motifsin the variable region might have a positive or negative impact on theantibody activity and is recognized to be a cause of antibodyheterogeneity. The presence of a glycan on the light chain of HMB3210was assessed on a reducing SDS-PAGE gel following incubation in thepresence or absence of the N-glycosidase PNG-ase F (FIG. 8). Thisanalysis indicated that a minority of the light chain is indeedglycosylated. The N113 residue was then removed and the correspondinggermline-encoded serine residue was restored. In parallel, anothersomatic mutation in framework-1 region of the light chain (P7T) was alsoremoved to restore the germline-encoded proline residue at position 7(IMGT numbering in the corresponding IGLV1-40*02 gene). A new HMB3210variant (named HMB3210v3) made by HMB3210 heavy chain VH.2 (SEQ ID 17)and HMB3210 light chain VL.3 was then produced and tested for itsneutralizing activity against RSV A2 and MPV I-PV 03/01-6621 strains inparallel with the HMB3210v2 (made by HMB3210 heavy chain VH.2 (SEQ IDNO: 17) and light chain VL SEQ ID NO: 14). This variant (HMB3210v3)showed a slightly improved neutralization against both RSV and MPVstrains tested (FIG. 9), thus showing that the removal of the lightchain glycosylation site does not affect binding to RSV and MPV targetepitopes. The two antibody variants were then tested in parallel againsta panel of RVS and MPV viruses and shown to have comparable activitiesagainst all viruses tested (FIG. 10). In conclusion, HMB3210v3 is notglycosylated in the variable light chain and is overall poorly mutatedas compared to the germline heavy and light chain genes having only 8amino acid somatic mutations in the heavy chain and 4 in the lightchain: S58A (HCDR2), 1655 (HCDR2), Y66D (HFR3), V71A (HFR3), N85T(HFR3), Y88F (HFR3), V101I (HFR3), Y103F (HFR3), G56D (LCDR2), S65N(LCDR2), G78A (LFR3) and S109R (LCDR3) (all positions were indicatedaccording to the IMGT numbering).

Example 9. HMB3210 and HMB2430 Cross-Reactivity with MPV Relies onSomatic Mutations

In order to gain insights into the role of somatic mutations in thecross-reactivity of HMB2430 and HMB3210 against RSV and MPV, thegermlined versions of both mAbs were synthesized and tested for theircapacity to neutralize RSV A2 and MPV I-PV 03/01-6621 strains. BothHMB2430 and HMB3210 germlined mAbs (HMB2430-GL and HMB3210-GL,respectively) were made of VH.3 and VL.2 in case of HMB2430-GL and ofVH.3 and VL.4 in case of HMB3210-GL. Both germlined forms of the mAbsefficiently neutralized RSV to levels comparable to those observed withthe original somatically mutated HMB3210 and HMB2430 (FIG. 11). However,HMB3210-GL and HMB2430-GL failed to neutralize MPV, thus indicating thatsomatic mutations are indispensable for MPV neutralization. To furtherunderstand whether somatic mutations of heavy or light chain are bothresponsible for neutralization of MPV, we produced antibodies carryingeither the heavy or the light chain in the germline configuration:HMB2430-VHGL-VLSM made by VH.3 and VL; HMB2430-VHSM-VLGL made by VH.2and VL.2; HMB3210-VHGL-VLSM made by VH.3 and VL; HMB3210-VHSM-VLGL madeby VH.2 and VL.4. Removal of somatic mutations in the heavy chain ofHMB3210 did not affect neutralization of RSV or MPV viruses, whileremoval of somatic mutations in the heavy chain of HMB2430 affectedneutralization of MPV, albeit maintaining neutralization of RSV. Removalof somatic mutations in the light chain of both HMB2430 and HMB3210abolished MPV neutralization, while not affecting RSV neutralization(FIG. 11). Taken together, these findings indicate that HMB3210 andHMB2430 were initially selected by RSV and subsequently developed,through the accumulation of somatic mutations, cross-reactivity againstMPV. Overall only 3 somatic mutations in the light chain CDRs accountfor the acquisition of MPV cross-reactivity in a RSV-specific germlinedantibody. Of note, HMB2430 and HMB3210 (not clonally related) share thesame somatic mutation in LCDR3 S to R at position 109.

Example 10. HMB3210 Recognition of Pre- and Post-Fusion F ProteinConformations

A DNA construct encoding RSV F residues 26-136 and 147-512(corresponding to the F ectodomain without the fusion peptide of the RSVstrain A2) with a C-terminal histidine tag was codon optimized andsynthesized. Recombinant F was expressed using a baculovirus expressionvector in Sf21 cells and purified from the supernatant by nickelaffinity and size exclusion chromatography (SEC). A similar constructwas already used by others (Swanson et al. PNAS 2011 and McLellan et al.J Virol 2011) to solve the crystal structure of the post-fusion Fprotein. The protein was analyzed under non-reducing conditions on anSDS-PAGE gel and gave a band at kDa and when analyzed by SEC on a 5200column the “post-fusion” RSV F protein eluted as a symmetric peak withan apparent molecular weight of ≈150 kDa that corresponds to the MW ofthe trimeric F protein and overlaps with the elution volume of humanIgG1 antibodies. The “post-fusion” F protein was incubated with eitherHMB3210 or Palivizumab and the two mixtures were run on a 5200 column.The incubation of the “post-fusion” F protein with Palivizumab shiftedthe elution peak to a lower elution volume (corresponding to an apparentMW of X300 kDa) as compared to the F protein alone indicating thatPalivizumab bound to the “post-fusion” F protein, as already reported.Of note, the incubation of HMB3210 with “post-fusion” F protein did notresult in a shifting of the elution volume (FIG. 12). The fact thatHMB3210 and the “post-fusion” F protein elute as independent moleculesindicates that HMB3210, unlike Palivizumab, does not bind the“post-fusion” F protein.

A stabilized form of the full-length pre-fusion RSV F protein was thensynthesized following the strategy adopted by Magro et al. PNAS 2012 bysubstituting the 4 amino acid residues (L481, D489, 5509 and D510) withcysteines and by substituting the 9 basic amino acid residues (R106,R108, R109, K131, K132, R133, K134, R135 and R136) at the two F proteincleavage sites with N residues to ablate the furine cleavage sites. TheF protein sequence was additionally modified by the insertion of a TEVcleavage site after the transmembrane region, followed by GFP and a6-His tag at the C-terminus to facilitate purification. The 4 introducedcysteines were positioned, according to the PIV-5 pre-fusion Fstructure, in a way that the formation of inter-monomeric disulphidebonds was possible only when the F protein was in the pre-fusionconformation, but not when refolded into the post-fusion structure, thusenabling the stabilization of the pre-fusion F protein. The stabilizedpre-fusion F protein was described by Magro et al. to be heterogenous,since it also contains a proportion of molecules in which the additionalcysteine residues were not disulphide bonded. The F protein constructhere described was produced using a baculovirus expression vector inSf21 cells, solubilized from cell membranes with a mild detergent andpurified by nickel affinity and size exclusion chromatography. Thepurified pre-fusion F protein was analyzed by SEC on a S200 column andeluted as a symmetric peak with an apparent molecular weight of ≈150 kDathat correspond to the MW of the trimeric F protein and that overlapswith the elution volume of human IgG1. The incubation of the pre-fusionF protein with Palivizumab shifted the elution peak to a lower elutionvolume (corresponding to an apparent MW of ≈300 kDa) as compared to theF protein alone and also induced the formation of a high molecularweight complex that eluted in the void volume of the column that mightbe related to the formation of larger aggregates. A similar shift in theelution volume was also observed when HMB3210v2 was incubated with thepre-fusion protein (FIG. 13). These results indicate that Palivizumabbind to both the pre-fusion and post-fusion forms of the F protein,while HMB3210 selectively recognizes the pre-fusion form of the Fprotein. The two F proteins (pre- and post-fusion forms) were alsotested by surface plasmon resonance (SPR). Palivizumab bound to bothpre- and post-fusion proteins with similar affinities, while HMB3210v3selectively bound to the pre-fusion F protein with high affinity (Kdconstant of 0.1 nM as compared to the Palivizumab Kd of 2 nM) (FIG. 14).

Example 11. HMB3210v3 Cross-Neutralizes the Two Animal ParamyxovirusesBovine Respiratory Syncytial Virus (BRSV) and Pneumonia Virus of Mice(PVM)

The breadth of reactivity of HMB3210 was also assessed on two otheranimal paramyxoviruses: BRSV and PVM, two viruses that share with RSV81% and 40% amino acid identity in the F protein, respectively. HMB3210was tested for its ability to neutralize PVM strain 15 and BRSV strainRB94 and shown to be effective against these viruses with IC50 values of100 ng/ml and 10 ng/ml, respectively. These results indicate that inaddition to the human paramyxoviruses RSV and MPV, HMB3210 is alsoeffective against other two viruses of the paramyxoviridae family.

Example 12. Inhibition of Virus Spreading by HMB3210

We also measured the ability of HMB3210 and the D25 RSV-specificantibody to prevent cell-to-cell viral spread, which has been reportedto be a distinct property of anti-RSV antibodies independent of theneutralizing activity. We infected Hep-2 cells with RSV A or B strains,added after 20 hours different concentrations of antibodies and examinedthe formation of syncytia on day 3 to determine the 50% antibodyconcentration inhibiting viral spread, here defined as IS50. Bothantibodies were capable of inhibiting viral spread, but at higherconcentrations. Interestingly, in this assay HMB3210 showed IS50comparable to those of the more potent neutralizing antibody D25 (FIG.15).

Example 13. Prophylactic and Therapeutic Efficacy of HMB3210 AgainstRSV, MPV and PVM

In the RSV mouse models HMB3210 was on average five to ten fold morepotent than Palivizumab in reducing RSV lung titers and was effective atconcentrations as low as 0.12 mg/kg (FIG. 16A). In the MPV mouse modelHMB3210 was comparably effective (FIG. 16B). To test the therapeuticpotential of HMB3210 we infected STAT1-deficient mice with RSV andadministered HMB3210 on day 1, 2 or 3 post-infection. In spite of thelimitation of this model, due to the poor replication of the virus,HMB3210 showed therapeutic efficacy at all time points and reduced viraltiters and inflammatory cytokines in the lungs, (FIG. 17).

To test HMB3210 in a more relevant animal model of acute lowerrespiratory tract infection, we exploited its cross-reactivity with PVM,a virus that causes a lethal disease in mice following a very lowinoculum and recapitulates the features of severe RSV and MPV infectionin humans. In a prophylactic setting, HMB3210 fully protected mice fromlethality at 0.12 mg/kg and from body weight loss at 0.6 mg/kg (FIG.18A). Furthermore, in a therapeutic setting HMB3210 completely protectedfrom lethality when administered up to 3 days after infection both at 30and 5 mg/kg and conferred significant protection when given on day 4 or5 at 30 mg/kg (FIG. 18B-18D). In this system Ribavirin, which is theonly approved standard of care for therapy in humans, as previouslydescribed (Bonville et al., 2004, Journal of Virology 78:7984-7989) wasineffective. Importantly, therapeutic delivery of HMB3210 efficientlyblocked further increase in lung viral RNA (FIG. 19). To address therole of effector mechanisms in vivo we compared the IgG1 HMB3210 with amutant that lacks complement and Fc receptor binding (HMB3210-LALA). Thetwo antibodies were compared for in vitro neutralizing activity andshown to be equivalent. When administered in limiting amounts in aprophylactic setting (0.12 mg/kg) HMB3210-LALA showed a severely reducedefficacy (FIG. 20A). In contrast, when used in a therapeutic settingHMB3210-LALA was as effective as HMB3210 at all doses tested (FIG. 20B).The therapeutic efficacy of HMB3210 in the PVM infection model, whereRibavirin is not effective, may be due to a combination of factors, suchas the potent neutralizing and spreading inhibition activity, theselective recognition of the pre-fusion protein which avoids theconsumption of the antibody by the abundant post-fusion proteins actingas decoys, and the failure to select escape mutants. Surprisingly, thetherapeutic efficacy of HMB3210 in the PVM mouse model does not requireeffector functions, suggesting that the antibody activity in vivo reliesprimarily on viral neutralization and inhibition of viral spread.

Example 14. HMB3210 Binds to a Highly Conserved Beta Strand on thePre-Fusion F Protein, which is not Accessible on the Post-Fusion FProtein

To identify the epitope recognized by HMB3210 we screened a library of7,095 structured peptides covering the full sequence of human RSV Fprotein. Experiments shown in example 5 showed that HMB3210 reacted inWestern blots with RSV and MPV F proteins under non-reducing conditions,suggesting that HMB3210 target an epitope whose conformation is stablein the presence of the anionic detergent SDS (FIGS. 5 and 6). Thelibrary screening approach led to the identification of a putativeHMB3210 epitope in the N-terminal region of F2, spanning residuesSAVSKGYLSALRTGWYTSVIT (SEQ ID NO: 63). The core sequence in this region,YLSALRTGW (SEQ ID NO: 64), is highly conserved between RSV, MPV, BRSVand PVM (FIG. 21) where the variants YLSVLRTGW (SEQ ID NO: 65),YFSALRTGW (SEQ ID NO: 66), YFSVLRTGW (SEQ ID NO: 67), YKSALRTGW (SEQ IDNO: 68) and YKSVLRTGW (SEQ ID NO: 69) are also recognized by HMB3210.This sequence is not exposed on the surface of the post-fusion RSV Fprotein, but, in a model of the RSV pre-fusion F protein built aroundthe PIVS F protein structure (Yin, et al., 2006, Nature 439:38-44), isexpected to locate in an exposed beta-strand in proximity to the loopregion targeted by Palivizumab (FIG. 22). This mapping is consistentwith the specificity of HMB3210 for the pre-fusion F protein shown inexample 10. This epitope is close, but distinct from that recognized byPalivizumab and is centered around the YLSVLRTGW sequence which ishighly conserved amongst 551 virus strains comprising 364 HRSV, 162HMPV, 8 BRSV and 5 PVM strains (FIG. 23).

1.-32. (canceled)
 33. A method of producing an antibody or an antigenbinding fragment thereof, the method comprising culturing a recombinanthost cell that comprises (a) a polynucleotide encoding the antibody orantigen binding fragment or (b) a polynucleotide encoding a heavy chainpolypeptide of the antibody or antigen binding fragment and apolynucleotide encoding a light chain polypeptide of the antibody orantigen binding fragment, for a time and under conditions sufficient forexpression of the antibody or antigen binding fragment, wherein theantibody or antigen binding fragment comprises: (i) a CDRH1 having theamino acid sequence of SEQ ID NO.:1, a CDRH2 having the amino acidsequence of SEQ ID NO.:2 or SEQ ID NO.:39, a CDRH3 having the amino acidsequence of SEQ ID NO.:3 or SEQ ID NO.: 40, a CDRL1 having the aminoacid sequence of SEQ ID NO.:4, a CDRL2 having the amino acid sequence ofSEQ ID NO.:5 or SEQ ID NO.:41, and a CDRL3 having the amino acidsequence of SEQ ID NO.:6, SEQ ID NO.:35, or SEQ ID NO.:42; or (ii) aCDRH1 having the amino acid sequence of SEQ ID NO.:1 or SEQ ID NO.:19, aCDRH2 having the amino acid sequence of SEQ ID NO.:2, SEQ ID NO.:20, orSEQ ID NO.:39, a CDRH3 having the amino acid sequence of SEQ ID NO.:3,SEQ ID NO.:21, or SEQ ID NO.: 53, a CDRL1 having the amino acid sequenceof SEQ ID NO.:4, a CDRL2 having the amino acid sequence of SEQ ID NO.:22or SEQ ID NO.:41, and a CDRL3 having the amino acid sequence of SEQ IDNO.:23 or SEQ ID NO.:54, wherein the antibody or antigen bindingfragment is capable of specifically binding to the pre-fusion F proteinand not the post-fusion F protein of Respiratory Syncytial Virus (RSV)and metapneumovirus (MPV).
 34. The method of claim 33, wherein therecombinant host cell comprises a eukaryotic cell.
 35. The method ofclaim 33, wherein the recombinant host cell comprises a yeast cell, ananimal cell, or a plant cell.
 36. The method of claim 33, wherein therecombinant host cell comprises a mammalian cell.
 37. The method ofclaim 33, wherein the recombinant host cell comprises a human cell, aCHO cell, a HEK293T cell, a PER.C6 cell, a NS0 cell, a myeloma cell, ora hybridoma cell.
 38. The method of claim 37, wherein the recombinanthost cell comprises a CHO cell.
 39. The method of claim 33, furthercomprising isolating the antibody or antigen binding fragment.
 40. Themethod of claim 39, further comprising purifying the antibody or antigenbinding fragment.
 41. The method of claim 33, wherein the polynucleotideof (a) or one or both of the polynucleotides of (b) is comprised in anexpression vector.
 42. The method of claim 33, wherein the antibody orantigen binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3 amino acid sequences of SEQ ID NOs.: (i) 1-6, respectively;(ii) 1, 2, 3, 4, 5, and 35, respectively; (iii) 1, 39, 40, 4, 41, and42, respectively; (iv) 1, 39, 40, 4, 5, and 6, respectively; (v) 1, 2,3, 4, 41, and 42, respectively; (vi) 19, 20, 21, 4, 22, and 23,respectively; (vii) 1, 39, 53, 4, 41, and 54, respectively; (viii) 1,39, 53, 4, 22, and 23, respectively; or (ix) 19, 20, 21, 4, 41, and 54,respectively.
 43. The method of claim 33, wherein the antibody orantigen binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3 amino acid sequences of SEQ ID NOs.: 1, 2, 3, 4, 5, and 35,respectively.
 44. The method of claim 33, wherein the antibody orantigen binding fragment comprises: (i) a VH having at least 80%identity to the amino acid sequence of SEQ ID NO.:13, SEQ ID NO.:17, orSEQ ID NO.:49, and a VL having at least 80% identity to the amino acidsequence of SEQ ID NO.:14, SEQ ID NO.:37, or SEQ ID NO.:50; or (ii) a VHhaving at least 80% identity to the amino acid sequence of SEQ IDNO.:29, SEQ ID NO.:33, or SEQ ID NO.:59, and a VL having at least 80%identity to the amino acid sequence of SEQ ID NO.:30 or SEQ ID NO.:60.45. The method of claim 33, wherein the antibody or antigen bindingfragment comprises: (i) a VH having at least 90% identity to the aminoacid sequence of SEQ ID NO.:13, SEQ ID NO.:17, or SEQ ID NO.:49, and aVL having at least 90% identity to the amino acid sequence of SEQ IDNO.:14, SEQ ID NO.:37, or SEQ ID NO.:50; or (ii) a VH having at least90% identity to the amino acid sequence of SEQ ID NO.:29, SEQ ID NO.:33,or SEQ ID NO.:59, and a VL having at least 90% identity to the aminoacid sequence of SEQ ID NO.:30 or SEQ ID NO.:60.
 46. The method of claim33, wherein the antibody or antigen binding fragment comprises a VH anda VL having the amino acid sequences of SEQ ID NOs.: (i) 13 and 14,respectively; (ii) 17 and 14, respectively; (iii) 17 and 37,respectively; (iv) 49 and 50, respectively; (v) 49 and 14, respectively;(vi) 17 and 50, respectively; (vii) 29 and 30, respectively; (viii) 33and 30, respectively; (ix) 59 and 60, respectively; (x) 59 and 30,respectively; or (xi) 33 and 60, respectively.
 47. The method of claim33, wherein the antibody or antigen binding fragment comprises a VHhaving the amino acid sequence of SEQ ID NO.:17 and a VL having theamino acid sequence of SEQ ID NO.:
 37. 48. The method of claim 33,wherein the antibody or antigen binding fragment is capable ofneutralizing infection of RSV, MPV, and PVM.
 49. The method of claim 48,wherein the concentration of the antibody or antigen binding fragmentrequired for 50% neutralization of RSV, MPV or PVM is 500 ng/mL or less.50. The method of claim 33, wherein the antibody or antigen bindingfragment is capable of neutralizing infection of both group A and groupB RSV as well as both group A and group B MPV.
 51. The method of claim33, wherein the antibody or antigen binding fragment is capable ofneutralizing infection of MPV subgroups A1, A2, B1 and B2.
 52. Themethod of claim 33, wherein the antibody is HMB3210 variant 3, HMB3210variant 1, HMB3210 variant 2, HMB3210 variant 4, HMB3210 variant 5,HMB3210 variant 6, HMB2430 variant 1, HMB2430 variant 2, HMB2430 variant3, HMB2430 variant 4 or HMB2430 variant 5.